Control of plant stress tolerance, water use efficiency and gene expression using novel aba receptor proteins and synthetic agonists

ABSTRACT

The present invention provides methods of regulating plant stress tolerance.

CROSS-REFERENCE TO RELATED PATENT APPLICATIONS

This application is a divisional of application Ser. No. 12/705,413,filed Feb. 12, 2010, which claims benefit of priority to U.S.Provisional Patent Application No. 61/207,684, filed Feb. 13, 2009, eachof which is incorporated by reference for all purposes.

BACKGROUND OF THE INVENTION

Abscisic acid (ABA) has been the focus of intense investigation since itwas identified in the 1960s as an endogenous small molecule growthinhibitor and regulator of plant stress physiology (K. Ohkuma, J. L.Lyon, F. T. Addicott, O. E. Smith, Science 142, 1592 (1963); C. F.Eagles, P. E. Wareing, Physiologia Plantarum 17, 697 (1964); J. W.Cornforth, B. V. Milborrow, G. Ryback, Nature 206, 715 (1965); J. W.Cornforth, B. V. Milborrow, G. Ryback, P. F. Wareing, Nature 205, 1269(1965); D. Imber, M. Tal, Science 169 592 (1970)). Indeed, when oneincreases plant ABA sensitivity, improved drought and other stresstolerance results. See, e.g. Wang et al., Plant J. 43:413-424 (2005);Pei et al. Science 282:287-290 (1998); US Patent Publication No2004/0010821. Genetic analyses have identified many factors involved inABA signaling, including the type 2 C protein phosphatases (PP2Cs) ABI1,ABI2 and relatives that form the closely related ABI1/AHG1 clades thatfunction as redundant negative regulators of ABA signaling (R. R.Finkelstein, S. S. L. Gampala, C. D. Rock, The Plant Cell 14, S15(2002); P. McCourt, Annual Review of Plant Physiology and PlantMolecular Biology 50, 219 (1999); A. Schweighofer, H. Hirt, I. Meskiene,Trends in Plant Science 9, 236 (2004)). Several ABA binding proteinshave been reported, however it is not clear how they regulate the myriadeffects of ABA, because they do not appear to act through knownregulators of its signaling pathway (X. Liu et al., Science 315, 1712(Mar. 23, 2007); F. A. Razem, A. El-Kereamy, S. R. Abrams, R. D. Hill,Nature 439, 290 (2006); Y. Y. Shen et al., Nature 443, 823 (Oct. 19,2006)). Additionally, the characterized receptors show negligiblebinding to the non-natural stereoisomer (−)-ABA 1 at concentrations˜1000-fold higher than their K_(d)s for (+)-ABA 2. (−)-ABA is bioactivein most ABA assays (B.-L. Lin, H.-J. Wang, J.-S. Wang, L. I. Zaharia, S.R. Abrams, Journal of Experimental Botany 56, 2935 (2005); D. Huang etal., The Plant Journal 50, 414 (2007)) and acts through the samesignaling pathway as (+)-ABA (E. Nambara et al., Genetics 161, 1247(July 2002)), suggesting that receptors that recognize both (−) and(+)-ABA remain to be discovered.

BRIEF SUMMARY OF THE INVENTION

The present invention provides for plants (or a plant cell, seed,flower, leaf, fruit, or other plant part from such plants) comprising aheterologous expression cassette, the expression cassette comprising apromoter operably linked to a polynucleotide encoding a PYR/PYL receptorpolypeptide, wherein the plant has improved stress tolerance compared toa plant lacking the expression cassette.

In some embodiments, the PYR/PYL receptor polypeptide comprises one ormore of SEQ ID NOs:1, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102,103, 104, 105, 106, 107 and/or 138.

In some embodiments, the PYR/PYL receptor polypeptide is at least 70%(e.g., at least 70%, 80%, 90%, 95%) identical to any of SEQ ID NOs:2-90or 108-137.

In some embodiments, the PYR/PYL receptor polypeptide is aconstitutively-active form such that the receptor will bind a type 2protein phosphatase (PP2C) in a yeast two-hybrid assay in the absence ofabscisic acid or an ABA agonist.

In some embodiments, the PYR/PYL receptor polypeptide bind a type 2protein phosphatase (PP2C) in a yeast two-hybrid assay in the presence,but not in the absence, of abscisic acid or an ABA agonist.

In some embodiments, the plant has improved drought tolerance comparedto a plant lacking the expression cassette.

In some embodiments, the promoter is a root-specific promoter.

In some embodiments, the promoter is specific for an aerial portion ofthe plant.

In some embodiments, the promoter is inducible.

The present invention also provides for methods of increasing stresstolerance in a plant as described above. In some embodiments, the methodcomprises contacting the plant with a sufficient amount of a compound toincrease stress tolerance compared to not contacting the plant with thecompound, wherein the compound is selected from the following formulas:

-   -   wherein    -   R¹ is selected from the group consisting of aryl and heteroaryl,        optionally substituted with 1-3 R^(1a) groups;    -   each R^(1a) is independently selected from the group consisting        of H, halogen, C₁₋₆ alkyl, C₁₋₆ haloalkyl, C₂₋₆ alkenyl, C₂₋₆        alkynyl, C₁₋₆ alkoxy, C₁₋₆ haloalkoxy, C₁₋₆ hydroxyalkyl,        —NR′R″, —SR′, —OH, —CN, —NO₂, —C(O)R′, —C(O)OR′, —C(O)NR′R″,        —N(R′)C(O)R″, —N(R′)C(O)OR″, —N(R′)C(O)NR′R″, —OP(O)(OR′)₂,        —S(O)₂OR′, —S(O)₂NR′R″, cycloalkyl, heterocycloalkyl, aryl and        heteroaryl, wherein the aryl group is optionally substituted        with —NO₂ and the heteroaryl group is optionally substituted        with C₁₋₆ alkyl;    -   alternatively, adjacent R^(1a) groups can combine to form a        member selected from the group consisting of cycloalkyl,        heterocycloalkyl, aryl and heteroaryl, wherein the aryl group is        optionally substituted with —OH;    -   R′ and R″ are each independently selected from the group        consisting of H and C₁₋₆ alkyl;    -   R² is selected from the group consisting of C₂₋₆ alkenyl,        cycloalkenyl, aryl and heteroaryl;    -   R³ is H or is optionally combined with R² and the atoms to which        each is attached to form a heterocycloalkyl optionally        substituted with 1-3 R^(1a) groups;    -   R⁴ is a heteroaryl, optionally substituted with 1-3 R^(1a)        groups;    -   R⁵ is selected from the group consisting of C₁₋₆ alkyl and aryl,        wherein the aryl is optionally substituted with 1-3 R^(1a)        groups;    -   each of R⁶ and R⁷ are independently selected from the group        consisting of aryl and heteroaryl, each optionally substituted        with 1-3 R^(1a) groups;    -   R⁸ is selected from the group consisting of cycloalkyl and aryl,        each optionally substituted with 1-3 R^(1a) groups;    -   R⁹ is H or is optionally combined with a R^(1a) group of R⁸ and        the atoms to which each is attached to form a heterocycloalkyl;        subscript n is 0-2;    -   X is absent or is selected from the group consisting of —O—, and        —N(R′)—;    -   Y is absent or is selected from the group consisting of —C(O)—        and —C(R′,R″)—;    -   Z is absent or is selected from the group consisting of —N═, and        —C(S)—N(R′)—, such that one of Y and Z is absent;    -   each of R¹⁰ and R¹¹ are independently selected from the group        consisting of H, C₁₋₆ alkyl, —C(O)OR′, and C₁₋₆ alkenyl-C(O)OH,        wherein at least two of the R¹⁰ and R¹¹ groups are C₁₋₆ alkyl        and at least one of the R¹⁰ and R¹¹ groups is C₁₋₆        alkenyl-C(O)OH;    -   alternatively, two R¹⁰ or R¹¹ groups attached to the same carbon        are combined to form ═O;    -   alternatively, one R¹⁰ group and one R¹¹ group are combined to        form a cycloalkyl having from 3 to 6 ring members;    -   each of subscripts k and m is an integer from 1 to 3, such that        the sum of k and m is from 3 to 4;    -   each of subscripts p and r is an integer from 1 to 10;    -   wherein two of the R¹⁰ and R¹¹ groups on adjacent carbons are        combined to form a bond;    -   R¹² is a C₁₋₆ alkyl, substituted with a ═O;    -   R¹³ is C₁₋₆ alkenyl-C(O)OH;    -   R¹⁴ is selected from the group consisting of H and C₁₋₆ alkyl;        and    -   subscript r is an integer from 1 to 10;        with the proviso that when R¹ is 4-bromo-naphthalen-1-yl, and n        is 1, R² is other than unsubstituted pyrid-2-yl

The present invention also provides an expression cassette comprising apromoter operably linked to a polynucleotide encoding a PYR/PYL receptorpolypeptide, wherein introduction of the expression cassette into aplant results in the plant having improved stress tolerance compared toa plant lacking the expression cassette.

In some embodiments, the PYR/PYL receptor polypeptide comprises one ormore of SEQ ID NOs:1, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102,103, 104, 105, 106, 107 and/or 138.

In some embodiments, the PYR/PYL receptor polypeptide is at least 70%(e.g., at least 70%, 80%, 90%, 95%) identical to any of SEQ ID NOs:2-90or 108-137.

In some embodiments, the PYR/PYL receptor polypeptide is aconstitutively-active form such that the receptor will bind a type 2protein phosphatase (PP2C) in a yeast two-hybrid assay in the absence ofabscisic acid or an ABA agonist.

In some embodiments, the PYR/PYL receptor polypeptide bind a type 2protein phosphatase (PP2C) in a yeast two-hybrid assay in the presence,but not in the absence, of abscisic acid or an ABA agonist.

In some embodiments, the plant has improved drought tolerance comparedto a plant lacking the expression cassette.

In some embodiments, the promoter is a root-specific promoter. In someembodiments, the promoter is specific for an aerial portion of theplant. In some embodiments, the promoter is inducible.

The present invention also provides for expression vectors comprising anexpression cassette of the invention (e.g., as described above).

The present invention also provides for methods of making a plant withincreased stress tolerance. In some embodiments, the method comprises:

introducing the an expression cassette of the invention (e.g., asdescribed above) into a plurality of plants; andselecting a plant comprising the expression cassette having increasedstress tolerance compared to a plant lacking the expression cassette.

The present invention also provides an agricultural chemical formulationformulated for contacting to plants, the formulation comprising acompound selected from the following formulas:

-   -   wherein    -   R¹ is selected from the group consisting of aryl and heteroaryl,        optionally substituted with 1-3 R^(1a) groups;    -   each R^(1a) is independently selected from the group consisting        of H, halogen, C₁₋₆ alkyl, C₁₋₆ haloalkyl, C₂₋₆ alkenyl, C₂₋₆        alkynyl, C₁₋₆ alkoxy, C₁₋₆ haloalkoxy, C₁₋₆ hydroxyalkyl,        —NR′R″, —SR′, —OH, —CN, —NO₂, —C(O)R′, —C(O)OR′, —C(O)NR′R″,        —N(R′)C(O)R″, —N(R′)C(O)OR″, —N(R′)C(O)NR′R″, —OP(O)(OR′)₂,        —S(O)₂OR′, —S(O)₂NR′R″, cycloalkyl, heterocycloalkyl, aryl and        heteroaryl, wherein the aryl group is optionally substituted        with —NO₂ and the heteroaryl group is optionally substituted        with C₁₋₆ alkyl;    -   alternatively, adjacent R^(1a) groups can combine to form a        member selected from the group consisting of cycloalkyl,        heterocycloalkyl, aryl and heteroaryl, wherein the aryl group is        optionally substituted with —OH;    -   R′ and R″ are each independently selected from the group        consisting of H and C₁₋₆ alkyl;    -   R² is selected from the group consisting of C₂₋₆ alkenyl,        cycloalkenyl, aryl and heteroaryl;    -   R³ is H or is optionally combined with R² and the atoms to which        each is attached to form a heterocycloalkyl optionally        substituted with 1-3 R^(1a) groups;    -   R⁴ is a heteroaryl, optionally substituted with 1-3 R^(1a)        groups;    -   R⁵ is selected from the group consisting of C₁₋₆ alkyl and aryl,        wherein the aryl is optionally substituted with 1-3 R^(1a)        groups;    -   each of R⁶ and R⁷ are independently selected from the group        consisting of aryl and heteroaryl, each optionally substituted        with 1-3 R^(1a) groups;    -   R⁸ is selected from the group consisting of cycloalkyl and aryl,        each optionally substituted with 1-3 R^(1a) groups;    -   R⁹ is H or is optionally combined with a R^(1a) group of R⁸ and        the atoms to which each is attached to form a heterocycloalkyl;        subscript n is 0-2;    -   X is absent or is selected from the group consisting of —O—, and        —N(R′)—;    -   Y is absent or is selected from the group consisting of —C(O)—        and —C(R′,R″)—;    -   Z is absent or is selected from the group consisting of —N═, and        —C(S)—N(R′)—, such that one of Y and Z is absent;    -   each of R¹⁰ and R¹¹ are independently selected from the group        consisting of H, C₁₋₆ alkyl, —C(O)OR′, and C₁₋₆ alkenyl-C(O)OH,        wherein at least two of the R¹⁰ and R¹¹ groups are C₁₋₆ alkyl        and at least one of the R¹⁰ and R¹¹ groups is C₁₋₆        alkenyl-C(O)OH;    -   alternatively, two R¹⁰ or R¹¹ groups attached to the same carbon        are combined to form ═O;    -   alternatively, one R¹⁰ group and one R¹¹ group are combined to        form a cycloalkyl having from 3 to 6 ring members;    -   each of subscripts k and m is an integer from 1 to 3, such that        the sum of k and m is from 3 to 4;    -   each of subscripts p and r is an integer from 1 to 10;    -   wherein two of the R¹⁰ and R¹¹ groups on adjacent carbons are        combined to form a bond;    -   R¹² is a C₁₋₆ alkyl, substituted with a ═O;    -   R¹³ is C₁₋₆ alkenyl-C(O)OH;    -   R¹⁴ is selected from the group consisting of H and C₁₋₆ alkyl;        and    -   subscript r is an integer from 1 to 10; with the proviso that        when R¹ is 4-bromo-naphthalen-1-yl, and n is 1, R² is other than        unsubstituted pyrid-2-yl

In some embodiments, the formulation further comprises at least one ofan herbicide, fungicide, pesticide, or fertilizer. In some embodiments,the formulation further comprises a surfactant.

The present invention also provides for a method of increasing stresstolerance in a plant, the method comprising contacting a plant with asufficient amount of a formulation as described above to increase stresstolerance in the plant compared to not contacting the plant with thecompound.

In some embodiments, the contacting step comprises delivering theformulation to the plant by aircraft or irrigation.

The present invention also provides for a method of identifying an agentthat agonizes a PYR/PYL polypeptide. In some embodiments, the methodcomprises

contacting one or more agents to a PYR/PYL polypeptide; anddetermining whether the one or more agents bind to and/or or activatethe PYR/PYL receptor polypeptide, wherein binding or activationidentifies the agent as an agonist of the PYR/PYL polypeptide.

In some embodiments, the determining step comprises contacting the agentto a cell comprising a two-hybrid system, wherein the two-hybrid systemsdetects interaction of the PYR/PYL polypeptide to a type 2 proteinphosphatase (PP2C), wherein agent-dependent interaction of the PYR/PYLpolypeptide to the PP2C identifies the agent as an agonist of thePYR/PYL polypeptide.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-1E. Pyrabactin is a seed selective ABA agonist. (A) Structuresof molecules described in this study. (B) Pyrabactin activity issuppressed by abi1-1. Seeds of the genotype shown at top were imbibed onmedia containing 25 μM pyrabactin and scored for germination 4 daysafter stratification. Shown at bottom are IC₅₀ values for pyrabactin'sgermination effect on the genotypes characterized. (C) Microarraycomparison of pyrabactin and ABA treatments in seeds. The Y-axis plotsthe log₂ transformed value for a probe's response to 25 μM pyrabactin(relative to untreated control) and the X-axis a probe's response to 1μM ABA. Plotted are data for probe sets that showed significantresponsiveness to ABA or pyrabactin, after removing germinationresponsive transcripts. (D) Microarray comparison of cycloheximide andABA responses in seeds. This plot shows the response of the same probesets analyzed in panel C, but the comparisons are to mRNAs fromcycloheximide treated seeds (y-axis). (E) Microarray comparison ofpyrabactin and ABA responses in seedlings. Seven-day old seedlings weretransferred to 10 μM ABA or 50 μM pyrabactin containing plates for 24hours and then mRNA samples profiled. Inset in each scatter plot is thePearson correlation coefficient for each comparison. Detailed microarraymethods are described in the Examples section.

FIGS. 2A-2E. PYR1 encodes an ABA responsive START-domain protein. (A)Pyr1 alleles. Shown are the allele names, strain names (in parentheses)and amino acid changes caused by the Pyr1 mutant alleles identified byscreening for pyrabactin resistant mutations. (B) Pictographicrepresentation of Pyr1 and Pyl1-Pyl4 expression values housed in publicmicroarray databases. The heatmap shown at top right is for the firstupper three panels and the bottom heatmap for the guard cell data. Plotswere made using the eFP browser (D. Winter et al., PLoS ONE 2, e718(2007)). (C) 35S::GFP-PYR1 complements pyr1-1. Seeds of the genotypesshown were stratified 4 days on 25 μM pyrabactin and then germinated atRT, 90% RH for 3 days in darkness. The Columbia wild type is unable togerminate under these conditions, but pyr1-1 does because it isresistant to pyrabactin. Introduction of a 35S::GFP-PYR1 construct intothe pyr1-1 genetic background restores pyrabactin sensitivity, whichindicates that the GFP fusion protein is functional. (D) Pyr/Pyls arerequired for normal ABA-induced gene expression in seedlings. Shown areqRT-PCR results for the ABA-responsive gene RD29. L, Ler; C, Col; and Q,quadruple mutant. (E) Pyr/Pyl genes are required for normal ABA-inducedstress-induced gene expression in seedlings. Shown are qRT-PCR resultsfor two ABA-responsive taqman probes, as described in the Examplessection. L=Ler, C=Col, Q=quadruple mutant.

FIGS. 3A-3C. ABA promotes PYR/PYL binding to PP2Cs. (A) Characterizationof the PYR/PYL protein interactions with HAB1. Shown are X-gal stains ofyeast colonies grown on plates containing the compounds shown at top.The Arabidopsis Genome Initiative (AGI) annotations for each PYR/PYLgene characterized is shown at the right of the panel. Not tested werePYL8 (AT5G53160) and PYL13 (AT4G18620). Each strain tested expresses anAD-HAB1 fusion protein and the BD-fusion shown at left. Chemicals weretested at 10 μM with the exception of epi-brassinolide (50 nM). (B) PYR1mutant proteins are defective in their interactions with HAR1. 3 PYR1amino acid substitution mutants that display strong pyrabactininsensitivity in Arabidopsis seeds were tested for their interactionswith HAB1 in the Y2H. (C) PYR1 interacts with ABI1 and ABI2 but not themutant protein encoded by abi2-1.

FIG. 4. GFP-PYR1 localizes to the cytoplasm and nucleoplasm. Confocalimages are shown of a 35S::GFP-PYR1 construct in the pyr1-1 mutantbackground. This construct complements the pyrabactin insensitivityphenotype of the pyr1-1 mutant.

FIGS. 5A-5B. Pyr1 and Pyl1, 2 and 4 function redundantly in ABAperception. (A) ABA responses in the triple and quadruple mutant linesare altered during germination. Seeds of the genotypes shown at top werestratified 4 days on media containing 0.9 μM (+)-ABA and thenphotographed 3 days after germination in darkness. The short hypocotylobserved in the quadruple mutant when germinated on (+)-ABA is due tothe presence of the erecta mutation that is tightly linked to the pyl2-1insertion allele. (B) ABA responses in the triple and quadruple mutantlines are altered during root growth. Seeds of the genotypes shown attop were stratified 4 days and then transferred to darkness (RT, 90%RH). After 30 hours, seeds with radicle emergence were transferred toplates contain 10 μM (+)-ABA and their roots photographed after anadditional 3 days growth in the dark.

FIGS. 6A-6C. PYR1 is an ABA receptor that regulates PP2C activity. (A)Reconstitution of ABA perception in vitro. Pull-down assays usingGST-HAB1 and 6×His-PYR1 (or mutants) were conducted using purifiedrecombinant proteins (left panel). GST-ABI1 and ABI2 were additionallytested in pull-downs using purified 6×His-PYR1 (or mutants) and crudelysates containing the PP2Cs shown. 10 μM (+)-ABA was used. (B) PYR1inhibits PP2C activity in the presence of ABA. The PP2C activity ofGST-HAB1 was tested in the presence or PYR1 or PYR1^(P88S) at differentconcentrations of ABA using the substrate pNPP. (C) ABA/PYL4-dependentinhibition of HAB1 PP2C activity. Recombinant PYL4 (refolded frominclusion bodies) and HAB1 were used in PP2C assays as described.Activity was measured for GST-HAB1 using the phosphatase substrate pNPP.Phosphatase initial reaction velocities were calculated in triplicate bymonitoring reactions over time using a plate reader in triplicate andused to calculate activities. The top panel shows the full concentrationranged studied; bottom panel a zoomed region of the lower concentrationstested. The specific activity of the GST-HAB1 used in these experimentswas 452.4±12.3 μmol/min/mg. Points plotted use ±SD as error bars.

FIG. 7. Proposed model for PYR/PYL control of ABA signaling. Withoutintending to limit the scope of the invention, we propose the followingmodel: In the absence of ABA (left), PYR/PYL proteins show low bindingto PP2Cs, and therefore, PP2C activity is high, which preventsphosphorylation and activation of SnRK2s and downstream factors (DFs).In the presence of ABA, PYR/PYLs bind and inhibit PP2Cs. This allowsaccumulation of phosphorylated downstream factors and ABAtranscriptional responses. The regulation of SnRK2s by PYR/PYLs may beindirect or may involve other factors.

FIG. 8. Activity of small molecule ABA agonists. This figure summarizesdata from screening small molecules for receptor activity of PYR1, PYL1,PYL2, PYL3, and PYL4.

FIG. 9. IC50 values for some compounds identified in the PP2C yeasttwo-hybrid assay. Compound numbers listed in left column correspond tocompounds identified in the assay summarized in FIG. 9. Compound 7653159corresponds to compound 7 in FIG. 9; compound 6655097 corresponds tocompound 6 in FIG. 9; and compound 7561035 corresponds to compound 9 inFIG. 9. For each compound, the ability of the compound to agonizePYR/PYL inhibition of the PP2C HAB1 was assessed using a phosphataseassay with the phosphatase substrate pNPP.

FIG. 10. Table of ABA-related phenotypes in the PYL4 overexpressionline. PYL4-overexpressing and pyr1; pyl1; pyl2; pyl4 quadruple mutantArabidopsis plants were examined for changes in stress responseassociated traits including flowering time, stature, chlorophyllcontent, and wiltiness relative to control Arabidopsis plants. Fulldetails for the construction of the mutant plants are provided in theExamples section.

FIGS. 11A-11E. Alignment of PYR1 and homologs from Arabidopsis. Thisfigure provides an alignment of Arabidopsis PYR/PYL protein sequences.The alignment displays, for example, absolutely conserved amino acids aswell as amino acids at positions that are typically conserved. Sequencesin the figure include the following PYR/PYL polypeptides: PYL12 (SEQ IDNO:77), PYL8 (SEQ ID NO:78), PYL7 (SEQ ID NO:79), PYL9 (SEQ ID NO:80),PYL11 (SEQ ID NO:81) PYL10 (SEQ ID NO:82), PYL13 (SEQ ID NO:83), PYL5(SEQ ID NO:84), PYL4 (SEQ ID NO:85), PYL6 (SEQ ID NO:86), PYL2 (SEQ IDNO:87), PYL3 (SEQ ID NO:88), PYR1 (SEQ ID NO:89), and PYL1 (SEQ IDNO:90). Consensus sequences derived from specified members are set forthbelow the alignment. ALL_Con=SEQ ID NOS:93-95; 1_(—)12_Con=SEQ IDNOS:96-99; 1_(—)6_Con=SEQ ID NOS:100, 139 and 102; 7_(—)10_Con=SEQ IDNOS:103 and 140; 11_(—)13_Con=SEQ ID NOS:106 and 107.

FIG. 12. Activity of additional ABA agonists. The listed compoundsinclude the naturally-occurring plant compound artemisinic acid, as wellas analogs thereof.

DEFINITIONS

The term “promoter,” as used herein, refers to a polynucleotide sequencecapable of driving transcription of a coding sequence in a cell. Thus,promoters used in the polynucleotide constructs of the invention includecis-acting transcriptional control elements and regulatory sequencesthat are involved in regulating or modulating the timing and/or rate oftranscription of a gene. For example, a promoter can be a cis-actingtranscriptional control element, including an enhancer, a promoter, atranscription terminator, an origin of replication, a chromosomalintegration sequence, 5′ and 3′ untranslated regions, or an intronicsequence, which are involved in transcriptional regulation. Thesecis-acting sequences typically interact with proteins or otherbiomolecules to carry out (turn on/off, regulate, modulate, etc.) genetranscription. A “plant promoter” is a promoter capable of initiatingtranscription in plant cells. A “constitutive promoter” is one that iscapable of initiating transcription in nearly all tissue types, whereasa “tissue-specific promoter” initiates transcription only in one or afew particular tissue types.

The term “plant” includes whole plants, shoot vegetative organs and/orstructures (e.g., leaves, stems and tubers), roots, flowers and floralorgans (e.g., bracts, sepals, petals, stamens, carpels, anthers), ovules(including egg and central cells), seed (including zygote, embryo,endosperm, and seed coat), fruit (e.g., the mature ovary), seedlings,plant tissue (e.g., vascular tissue, ground tissue, and the like), cells(e.g., guard cells, egg cells, trichomes and the like), and progeny ofsame. The class of plants that can be used in the method of theinvention is generally as broad as the class of higher and lower plantsamenable to transformation techniques, including angiosperms(monocotyledonous and dicotyledonous plants), gymnosperms, ferns, andmulticellular algae. It includes plants of a variety of ploidy levels,including aneuploid, polyploid, diploid, haploid, and hemizygous.

A polynucleotide sequence is “heterologous” to an organism or a secondpolynucleotide sequence if it originates from a foreign species, or, iffrom the same species, is modified from its original form. For example,when a promoter is said to be operably linked to a heterologous codingsequence, it means that the coding sequence is derived from one specieswhereas the promoter sequence is derived another, different species; or,if both are derived from the same species, the coding sequence is notnaturally associated with the promoter (e.g., is a geneticallyengineered coding sequence, e.g., from a different gene in the samespecies, or an allele from a different ecotype or variety).

A polynucleotide “exogenous” to an individual plant is a polynucleotidewhich is introduced into the plant by any means other than by a sexualcross. Examples of means by which this can be accomplished are describedbelow, and include Agrobacterium-mediated transformation, biolisticmethods, electroporation, and the like. Such a plant containing theexogenous nucleic acid is referred to here as a T₁ (e.g., in Arabidopsisby vacuum infiltration) or Ro (for plants regenerated from transformedcells in vitro) generation transgenic plant.

As used herein, the term “transgenic” describes a non-naturallyoccurring plant that contains a genome modified by man, wherein theplant includes in its genome an exogenous nucleic acid molecule, whichcan be derived from the same or a different plant species. The exogenousnucleic acid molecule can be a gene regulatory element such as apromoter, enhancer, or other regulatory element, or can contain a codingsequence, which can be linked to a heterologous gene regulatory element.Transgenic plants that arise from sexual cross or by selfing aredescendants of such a plant and are also considered “transgenic.”.

An “expression cassette” refers to a nucleic acid construct that, whenintroduced into a host cell, results in transcription and/or translationof an RNA or polypeptide, respectively. Antisense or sense constructsthat are not or cannot be translated are expressly included by thisdefinition. In the case of both expression of transgenes and suppressionof endogenous genes (e.g., by antisense, or sense suppression) one ofskill will recognize that the inserted polynucleotide sequence need notbe identical, but may be only “substantially identical” to a sequence ofthe gene from which it was derived. As explained below, thesesubstantially identical variants are specifically covered by referenceto a specific nucleic acid sequence.

“Increased” or “enhanced” PYR/PYL expression or activity refers to anaugmented change in the protein's expression or activity. Examples ofsuch increased activity or expression include, e.g., where PYR/PYLexpression is increased above control levels and/or where it isectopically expressed, e.g., in a place or time where it is notexpressed in a control. In some embodiments, PYR/PYL expression oractivity is increased above the level of that in wild-type,non-transgenic control plants (i.e., the quantity of PYR/PYL activity orexpression of the PYR/PYL gene is increased). In some embodiments,PYR/PYL expression or activity can be present, for example, in an organ,tissue, or cell where it is not normally detected in wild-type,non-transgenic control plants (i.e., PYR/PYL expression or activity isincreased within certain tissue types). In some embodiments, PYR/PYLexpression or activity is increased when its expression or activity ispresent in an organ, tissue or cell for a longer period than in awild-type, non-transgenic controls (i.e., duration of PYR/PYL expressionor activity is increased).

Two nucleic acid sequences or polypeptides are said to be “identical” ifthe sequence of nucleotides or amino acid residues, respectively, in thetwo sequences is the same when aligned for maximum correspondence asdescribed below. The terms “identical” or percent “identity,” in thecontext of two or more nucleic acids or polypeptide sequences, refer totwo or more sequences or subsequences that are the same or have aspecified percentage of amino acid residues or nucleotides that are thesame, when compared and aligned for maximum correspondence over acomparison window, as measured using one of the following sequencecomparison algorithms or by manual alignment and visual inspection. Whenpercentage of sequence identity is used in reference to proteins orpeptides, it is recognized that residue positions that are not identicaloften differ by conservative amino acid substitutions, where amino acidsresidues are substituted for other amino acid residues with similarchemical properties (e.g., charge or hydrophobicity) and therefore donot change the functional properties of the molecule. Where sequencesdiffer in conservative substitutions, the percent sequence identity maybe adjusted upwards to correct for the conservative nature of thesubstitution. Means for making this adjustment are well known to thoseof skill in the art. Typically this involves scoring a conservativesubstitution as a partial rather than a full mismatch, therebyincreasing the percentage sequence identity. Thus, for example, where anidentical amino acid is given a score of 1 and a non-conservativesubstitution is given a score of zero, a conservative substitution isgiven a score between zero and 1. The scoring of conservativesubstitutions is calculated according to, e.g., the algorithm of Meyers& Miller, Computer Applic. Biol. Sci. 4:11-17 (1988) e.g., asimplemented in the program PC/GENE (Intelligenetics, Mountain View,Calif., USA).

The phrase “substantially identical,” used in the context of two nucleicacids or polypeptides, refers to a sequence that has at least 25%sequence identity with a reference sequence. Alternatively, percentidentity can be any integer from 25% to 100%. Some embodiments includeat least: 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%,85%, 90%, 95%, or 99%, compared to a reference sequence using theprograms described herein; preferably BLAST using standard parameters,as described below. The present invention provides for nucleic acidsencoding polypeptides that are substantially identical to any of SEQ IDNO:2-90 or 108-137.

For sequence comparison, typically one sequence acts as a referencesequence, to which test sequences are compared. When using a sequencecomparison algorithm, test and reference sequences are entered into acomputer, subsequence coordinates are designated, if necessary, andsequence algorithm program parameters are designated. Default programparameters can be used, or alternative parameters can be designated. Thesequence comparison algorithm then calculates the percent sequenceidentities for the test sequences relative to the reference sequence,based on the program parameters.

A “comparison window”, as used herein, includes reference to a segmentof any one of the number of contiguous positions selected from the groupconsisting of from 20 to 600, usually about 50 to about 200, moreusually about 100 to about 150 in which a sequence may be compared to areference sequence of the same number of contiguous positions after thetwo sequences are optimally aligned. Methods of alignment of sequencesfor comparison are well-known in the art. Optimal alignment of sequencesfor comparison can be conducted, e.g., by the local homology algorithmof Smith & Waterman, Adv. Appl. Math. 2:482 (1981), by the homologyalignment algorithm of Needleman & Wunsch, J. Mol. Biol. 48:443 (1970),by the search for similarity method of Pearson & Lipman, Proc. Nat'l.Acad. Sci. USA 85:2444 (1988), by computerized implementations of thesealgorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin GeneticsSoftware Package, Genetics Computer Group, 575 Science Dr., Madison,Wis.), or by manual alignment and visual inspection.

Examples of algorithms that are suitable for determining percentsequence identity and sequence similarity are the BLAST and BLAST 2.0algorithms, which are described in Altschul et al. (1990) J. Mol. Biol.215: 403-410 and Altschul et al. (1977) Nucleic Acids Res. 25:3389-3402, respectively. Software for performing BLAST analyses ispublicly available through the National Center for BiotechnologyInformation (NCBI) web site. The algorithm involves first identifyinghigh scoring sequence pairs (HSPs) by identifying short words of lengthW in the query sequence, which either match or satisfy somepositive-valued threshold score T when aligned with a word of the samelength in a database sequence. T is referred to as the neighborhood wordscore threshold (Altschul et al, supra). These initial neighborhood wordhits acts as seeds for initiating searches to find longer HSPscontaining them. The word hits are then extended in both directionsalong each sequence for as far as the cumulative alignment score can beincreased. Cumulative scores are calculated using, for nucleotidesequences, the parameters M (reward score for a pair of matchingresidues; always >0) and N (penalty score for mismatching residues;always <0). For amino acid sequences, a scoring matrix is used tocalculate the cumulative score. Extension of the word hits in eachdirection are halted when: the cumulative alignment score falls off bythe quantity X from its maximum achieved value; the cumulative scoregoes to zero or below, due to the accumulation of one or morenegative-scoring residue alignments; or the end of either sequence isreached. The BLAST algorithm parameters W, T, and X determine thesensitivity and speed of the alignment. The BLASTN program (fornucleotide sequences) uses as defaults a word size (W) of 28, anexpectation (E) of 10, M=1, N=−2, and a comparison of both strands. Foramino acid sequences, the BLASTP program uses as defaults a word size(W) of 3, an expectation (E) of 10, and the BLOSUM62 scoring matrix (seeHenikoff & Henikoff, Proc. Natl. Acad. Sci. USA 89:10915 (1989)).

The BLAST algorithm also performs a statistical analysis of thesimilarity between two sequences (see, e.g., Karlin & Altschul, Proc.Nat'l. Acad. Sci. USA 90:5873-5787 (1993)). One measure of similarityprovided by the BLAST algorithm is the smallest sum probability (P(N)),which provides an indication of the probability by which a match betweentwo nucleotide or amino acid sequences would occur by chance. Forexample, a nucleic acid is considered similar to a reference sequence ifthe smallest sum probability in a comparison of the test nucleic acid tothe reference nucleic acid is less than about 0.01, more preferably lessthan about 10⁻⁵, and most preferably less than about 10⁻²⁰.

“Conservatively modified variants” applies to both amino acid andnucleic acid sequences. With respect to particular nucleic acidsequences, conservatively modified variants refers to those nucleicacids which encode identical or essentially identical amino acidsequences, or where the nucleic acid does not encode an amino acidsequence, to essentially identical sequences. Because of the degeneracyof the genetic code, a large number of functionally identical nucleicacids encode any given protein. For instance, the codons GCA, GCC, GCGand GCU all encode the amino acid alanine. Thus, at every position wherean alanine is specified by a codon, the codon can be altered to any ofthe corresponding codons described without altering the encodedpolypeptide. Such nucleic acid variations are “silent variations,” whichare one species of conservatively modified variations. Every nucleicacid sequence herein which encodes a polypeptide also describes everypossible silent variation of the nucleic acid. One of skill willrecognize that each codon in a nucleic acid (except AUG, which isordinarily the only codon for methionine) can be modified to yield afunctionally identical molecule. Accordingly, each silent variation of anucleic acid which encodes a polypeptide is implicit in each describedsequence.

As to amino acid sequences, one of skill will recognize that individualsubstitutions, in a nucleic acid, peptide, polypeptide, or proteinsequence which alters a single amino acid or a small percentage of aminoacids in the encoded sequence is a “conservatively modified variant”where the alteration results in the substitution of an amino acid with achemically similar amino acid. Conservative substitution tablesproviding functionally similar amino acids are well known in the art.

The following six groups each contain amino acids that are conservativesubstitutions for one another:

1) Alanine (A), Serine (S), Threonine (T);

2) Aspartic acid (D), Glutamic acid (E);

3) Asparagine (N), Glutamine (Q); 4) Arginine (R), Lysine (K); 5)Isoleucine (I), Leucine (L), Methionine (M), Valine (V); and 6)Phenylalanine (F), Tyrosine (Y), Tryptophan (W).

(see, e.g., Creighton, Proteins (1984)).

As used herein, the term “drought-resistance” or “drought-tolerance,”including any of their variations, refers to the ability of a plant torecover from periods of drought stress (i.e., little or no water for aperiod of days). Typically, the drought stress will be at least 5 daysand can be as long as, for example, 18 to 20 days or more (e.g., atleast 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 days),depending on, for example, the plant species.

DETAILED DESCRIPTION OF THE INVENTION I. Introduction

The present invention is based, in part, on the discovery of selectiveabscisic acid (ABA) agonist small organic molecules as well as aprotein, PYR1, which is required for the ABA agonist activity. It hasfurther been discovered that PYR1 is a member of the PYR/PYL receptorprotein family. Plants examined to date express more than one PYR/PYLreceptor protein family members and have at least somewhat redundantactivity. Increasing expression or activity of one or more PYR/PYLprotein in a plant therefore will result in increased ABA sensitivityand accordingly improved stress (e.g. cold, heat, salinity, or drought)response and tolerance as well as other desirable ABA-mediatedphenotypes.

Abscisic acid is a multifunctional phytohormone involved in a variety ofphyto-protective functions including bud dormancy, seed dormancy and/ormaturation, abscission of leaves and fruits, and response to a widevariety of biological stresses (e.g. cold, heat, salinity, and drought).ABA is also responsible for regulating stomatal closure by a mechanismindependent of CO₂ concentration. Thus, because PYR/PYL ABA receptorproteins mediate ABA signalling, these phenotypes can be modulated bymodulating expression of PYR/PYL. Phenotypes that are induced by ABA canbe increased or speeded in plants with increased expression of PYR/PYLwhereas such phenotypes can be reduced or slowed in plants withdecreased expression of PYR/PYL. PYR/PYL mediates ABA signaling as apositive regulator in, for example, seed germination, post-germinationgrowth, stomatal movement and plant tolerance to stress including, butnot limited to, drought. Accordingly, when abscisic acid sensitivity isincreased by overexpressing PYR/PYL, desirable characteristics in plantssuch as increased stress (e.g., drought) tolerance and delayed seedgermination is achieved. Other desirable characteristics that can begenerated in the plants of the invention include, e.g., a change inflowering time and/or increased chlorophyll content.

II. ABA Agonists

The present invention provides for small molecule ABA agonists, i.e.,compounds that activate PYR/PYL proteins. Exemplary ABA agonistsinclude, e.g., a compound selected from the following formulas:

-   -   wherein    -   R¹ is selected from the group consisting of aryl and heteroaryl,        optionally substituted with 1-3 R^(1a) groups;    -   each R^(1a) is independently selected from the group consisting        of H, halogen, C₁₋₆ alkyl, C₁₋₆ haloalkyl, C₂₋₆ alkenyl, C₂₋₆        alkynyl, C₁₋₆ alkoxy, C₁₋₆ haloalkoxy, C₁₋₆ hydroxyalkyl,        —NR′R″, —SR′, —OH, —CN, —NO₂, —C(O)R′, —C(O)OR′, —C(O)NR′R″,        —N(R′)C(O)R″, —N(R′)C(O)OR″, —N(R′)C(O)NR′R″, —OP(O)(OR′)₂,        —S(O)₂OR′, —S(O)₂NR′R″, cycloalkyl, heterocycloalkyl, aryl and        heteroaryl, wherein the aryl group is optionally substituted        with —NO₂ and the heteroaryl group is optionally substituted        with C₁₋₆ alkyl;    -   alternatively, adjacent R^(1a) groups can combine to form a        member selected from the group consisting of cycloalkyl,        heterocycloalkyl, aryl and heteroaryl, wherein the aryl group is        optionally substituted with —OH;    -   R′ and R″ are each independently selected from the group        consisting of H and C₁₋₆ alkyl;    -   R² is selected from the group consisting of C₂₋₆ alkenyl,        cycloalkenyl, aryl and heteroaryl;    -   R³ is H or is optionally combined with R² and the atoms to which        each is attached to form a heterocycloalkyl optionally        substituted with 1-3 R^(1a) groups;    -   R⁴ is a heteroaryl, optionally substituted with 1-3 R^(1a)        groups;    -   R⁵ is selected from the group consisting of C₁₋₆ alkyl and aryl,        wherein the aryl is optionally substituted with 1-3 R^(1a)        groups;    -   each of R⁶ and R⁷ are independently selected from the group        consisting of aryl and heteroaryl, each optionally substituted        with 1-3 R^(1a) groups;    -   R⁸ is selected from the group consisting of cycloalkyl and aryl,        each optionally substituted with 1-3 R^(1a) groups;    -   R⁹ is H or is optionally combined with a R^(1a) group of R⁸ and        the atoms to which each is attached to form a heterocycloalkyl;        subscript n is 0-2;    -   X is absent or is selected from the group consisting of —O—, and        —N(R′)—;    -   Y is absent or is selected from the group consisting of —C(O)—        and —C(R′,R″)—;    -   Z is absent or is selected from the group consisting of —N═, and        —C(S)—N(R′)—, such that one of Y and Z is absent;    -   each of R¹⁰ and R¹¹ are independently selected from the group        consisting of H, C₁₋₆ alkyl, —C(O)OR, and C₁₋₆ alkenyl-C(O)OH,        wherein at least two of the R¹⁰ and R¹¹ groups are C₁₋₆ alkyl        and at least one of the R¹⁰ and R¹¹ groups is C₁₋₆        alkenyl-C(O)OH;    -   alternatively, two R¹⁰ or R¹¹ groups attached to the same carbon        are combined to form ═O;    -   alternatively, one R¹⁰ group and one R¹¹ group are combined to        form a cycloalkyl having from 3 to 6 ring members;    -   each of subscripts k and m is an integer from 1 to 3, such that        the sum of k and m is from 3 to 4;    -   each of subscripts p and r is an integer from 1 to 10;    -   wherein two of the R¹⁰ and R¹¹ groups on adjacent carbons are        combined to form a bond;    -   R¹² is a C₁₋₆ alkyl, substituted with a ═O;    -   R¹³ is C₁₋₆ alkenyl-C(O)OH;    -   R¹⁴ is selected from the group consisting of H and C₁₋₆ alkyl;        and    -   subscript r is an integer from 1 to 10; with the proviso that        when R¹ is 4-bromo-naphthalen-1-yl, and n is 1, R² is other than        unsubstituted pyrid-2-yl

Exemplary compounds are further depicted in the Examples and Figures.See, e.g., FIGS. 9, 10, and 13.

The ABA agonist compounds of the present invention can be prepared by avariety of methods known to one of skill in the art. For example, thesulphonamide compounds can be prepared by reaction of a sulfonylchloride and an amine to provide the sulphonamide. Amide compounds ofthe present invention can be prepared in a similar fashion using an acidchloride in place of the sulfonyl chloride, or carbodiimide couplingreagents known to one of skill in the art. Additional methods of makingthe compounds of the present invention are known to one of skill in theart, for example, those described in Comprehensive OrganicTransformations, 2d ed., Richard C. Larock, 1999. The starting materialsfor the methods described above are commercially available(Sigma-Aldrich) or can be prepared by methods known to one of skill inthe art.

Phenotypes that are induced by ABA can be increased or speeded in plants(or plant parts such as seeds) by contacting the plants with asufficient amount of an ABA agonist of the invention to induce theABA-inducible phenotypes. ABA agonists of the invention are useful as,e.g., positive enhancers of, for example, delayed seed germination,post-germination growth, stomatal movement and plant tolerance to stressincluding, but not limited to, drought.

III. ABA Agonist Formulations

The present invention provides for agricultural chemical formulationformulated for contacting to plants, wherein the formulation comprisesan ABA agonist of the present invention. In some embodiments, the plantsthat are contacted with the agonists do not comprise or express aheterologous PYR/PYL polypeptide (e.g., the plants are not transgenic orare transgenic but express heterologous proteins other than heterologousPYR/PYL proteins). In some embodiments, the plants that are contactedwith the agonists do comprise or express a heterologous PYR/PYLpolypeptide as described herein.

The formulations can be suitable for treating plants or plantpropagation material, such as seeds, in accordance with the presentinvention, e.g., in a carrier. Suitable additives include bufferingagents, wetting agents, coating agents, polysaccharides, and abradingagents. Exemplary carriers include water, aqueous solutions, slurries,solids and dry powders (e.g., peat, wheat, bran, vermiculite, clay,pasteurized soil, many forms of calcium carbonate, dolomite, variousgrades of gypsum, bentonite and other clay minerals, rock phosphates andother phosphorous compounds, titanium dioxide, humus, talc, alginate andactivated charcoal. Any agriculturally suitable carrier known to oneskilled in the art would be acceptable and is contemplated for use inthe present invention. Optionally, the formulations can also include atleast one surfactant, herbicide, fungicide, pesticide, or fertilizer.

Treatment can be performed using a variety of known methods, e.g., byspraying, atomizing, dusting or scattering the compositions over thepropagation material or brushing or pouring or otherwise contacting thecompositions over the plant or, in the event of seed, by coating,encapsulating, or otherwise treating the seed. In an alternative todirectly treating a plant or seed before planting, the formulations ofthe invention can also be introduced into the soil or other media intowhich the seed is to be planted. In some embodiments, a carrier is alsoused in this embodiment. The carrier can be solid or liquid, as notedabove. In some embodiments peat is suspended in water as a carrier ofthe ABA agonist, and this mixture is sprayed into the soil or plantingmedia and/or over the seed as it is planted.

IV. Screening for New ABA Agonists and Antagonists

The present invention also provides methods of screening for ABAagonists and antagonists by screening for a molecule's ability to inducePYR/PYL-PP2C binding in the case of agonists, or to disrupt the abilityof ABA and other agonists to promote PYR/PYL-PP2C binding in the case ofantagonists. A number of different screening protocols can be utilizedto identify agents that agonize or antagonize a PYR/PYL polypeptide.

Screening can take place using isolated, purified or partially purifiedreagents. In some embodiments, purified or partially purified PYR/PYLpolypeptide can be used.

Alternatively, cell-based methods of screening can be used. For example,cells that naturally-express a PYR/PYL polypeptide or that recombinantlyexpress a PYR/PYL polypeptide can be used. In some embodiments, thecells used are plant cells, animal cells, bacterial cells, fungal cells,including but not limited to yeast cells, insect cells, or mammaliancells. In general terms, the screening methods involve screening aplurality of agents to identify an agent that modulates the activity ofa PYR/PYL polypeptide by, e.g., binding to PYR/PYL polypeptide, oractivating a PYR/PYL polypeptide or increasing expression of a PYR/PYLpolypeptide, or a transcript encoding a PYR/PYL polypeptide.

1. PYR/PYL Polypeptide Binding Assays

Optionally, preliminary screens can be conducted by screening for agentscapable of binding to a PYR/PRL polypeptide, as at least some of theagents so identified are likely PYR/PYL polypeptide modulators.

Binding assays can involve contacting a PYR/PYL polypeptide with one ormore test agents and allowing sufficient time for the protein and testagents to form a binding complex. Any binding complexes formed can bedetected using any of a number of established analytical techniques.Protein binding assays include, but are not limited to, methods thatmeasure co-precipitation or co-migration on non-denaturingSDS-polyacrylamide gels, and co-migration on Western blots (see, e.g.,Bennet, J. P. and Yamamura, H. I. (1985) “Neurotransmitter, Hormone orDrug Receptor Binding Methods,” in Neurotransmitter Receptor Binding(Yamamura, H. I., et al., eds.), pp. 61-89. Other binding assays involvethe use of mass spectrometry or NMR techniques to identify moleculesbound to PYR/PYL polypeptide or displacement of labeled substrates(e.g., labeled ABA). The PYR/PYL polypeptide protein utilized in suchassays can be naturally expressed, cloned or synthesized.

2. Activity

PYR/PYL polypeptide agonists can be identified by screening for agentsthat activate or increase activity of a PYR/PYL polypeptide. Antagonistscan be identified by reducing activity.

One activity assay involves testing whether a candidate agonist caninduce binding of a PYR/PYL protein to a type 2 protein phosphatase(PP2C) polypeptide in an agonist-specific fashion. Mammalian or yeasttwo-hybrid approaches (see, e.g., Bartel, P. L. et. al. Methods Enzymol,254:241 (1995)) can be used to identify polypeptides or other moleculesthat interact or bind when expressed together in a cell. In someembodiments, agents that agonize a PYR/PYL polypeptide are identified ina two-hybrid assay between a PYR/PYL polypeptide and a type 2 proteinphosphatase (PP2C) polypeptide, wherein an ABA agonist is identified asan agent that activates or enables binding of the PYR/PYL polypeptideand the PP2C polypeptide. Thus, the two polypeptides bind in thepresence, but not in the absence of the agent.

In some embodiments, agents that antagonize a PYR/PYL polypeptide areidentified in a two-hybrid assay between a PYR/PYL polypeptide and atype 2 protein phosphatase (PP2C) polypeptide, wherein an ABA antagonistis identified as an agent that decreases binding of the PYR/PYLpolypeptide and the PP2C polypeptide, optionally in the presence of ABAor a PYR/PYL ABA agonist. Thus, the antagonist blocks the normal bindingof the two polypeptides that is normally promoted by ABA or otheragonists, or alternatively, that is observed in constitutivelyinteracting PYR/PYL proteins.

3. Expression Assays

Screening for a compound that increases the expression of a PYR/PYLpolypeptide is also provided. Screening methods generally involveconducting cell-based or plant-based assays in which test compounds arecontacted with one or more cells expressing PYR/PYL polypeptide, andthen detecting an increase in PYR/PYL expression (either transcript ortranslation product). Assays can be performed with cells that naturallyexpress PYR/PYL or in cells recombinantly altered to express PYR/PYL, orin cells recombinantly altered to express a reporter gene under thecontrol of the PYR/PYL promoter.

Various controls can be conducted to ensure that an observed activity isauthentic including running parallel reactions with cells that lack thereporter construct or by not contacting a cell harboring the reporterconstruct with test compound.

4. Validation

Agents that are initially identified by any of the foregoing screeningmethods can be further tested to validate the apparent activity and/ordetermine other biological effects of the agent. In some cases, theidentified agent is tested for the ability to effect plant stress (e.g.,drought tolerance), seed germination, or another phenotype affected byABA. A number of such assays and phenotypes are known in the art and canbe employed according to the methods of the invention.

5. Solid Phase and Soluble High Throughput Assays

In the high throughput assays of the invention, it is possible to screenup to several thousand different modulators or ligands in a single day.In particular, each well of a microtiter plate can be used to run aseparate assay against a selected potential modulator, or, ifconcentration or incubation time effects are to be observed, every 5-10wells can test a single modulator. Thus, a single standard microtiterplate can assay about 100 (e.g., 96) modulators. If 1536 well plates areused, then a single plate can easily assay from about 100 to about 1500different compounds. It is possible to assay several different platesper day; assay screens for up to about 6,000-20,000 or more differentcompounds are possible using the integrated systems of the invention. Inaddition, microfluidic approaches to reagent manipulation can be used.

The molecule of interest (e.g., PYR/PYL or a cell expressing a PYR/PYLpolypeptide) can be bound to the solid state component, directly orindirectly, via covalent or non covalent linkage.

The invention provides in vitro assays for identifying, in a highthroughput format, compounds that can modulate the expression oractivity of PYR/PYL.

V. PYR/PYL Receptor Polypeptides

Polypeptides of the invention, when expressed in plants, mediate ABA andABA analog signaling. In some embodiments, the PYR/PYL polypeptidesinteract (e.g., in a yeast two-hybrid assay) with a PP2C polypeptide(e.g., ABI1 or 2 or orthologs thereof, e.g., from the group A subfamilyof PP2Cs) in an ABA, pyrabactin, or other ABA agonist—dependent manneras described herein.

A wide variety of PYR/PYL polypeptide sequences are known in the art andcan be used according to the methods and compositions of the invention.As noted herein, while PYR1 was originally identified as an ABA receptorin Arabidopsis, in fact PYR1 is a member of a group of at least 14proteins (PYR/PYL proteins) in the same protein family in Arabidopsisand that also mediate ABA signaling. This protein family is also presentin other plants (see, e.g., SEQUENCE LISTING) is characterized in partby the presence of one or more or all of a polyketide cyclase domain 2(PF10604), a polyketide cyclase domain 1 (PF03364), and a Bet V I domain(PF03364). START/Bet v 1 superfamily domain are described in, forexample, Radauer, B M C Evol. Biol. 8:286 (2008).

In situations where variants or orthologs of the above sequences aredesired, it can be useful to generate sequence alignments to identifyconserved amino acid or motifs (i.e., where alteration in sequences mayalter protein function) and regions where variation occurs in alignmentof sequences (i.e., where variation of sequence is not likely tosignificantly affect protein activity). SEQ ID NO:1, 91, and 92 provideconsensus sequences useful for identifying PYR/PYL polypeptides. Otheruseful consensus sequences include, e.g., EXLXXXDXXXXXXXXXXGGXHXL (SEQID NO:138); CxSxxxxxxxAPxxxxWxxxxxFxxPxxxxxFxxxC (SEQ ID NO:93),GxxRxVxxxSxxPAxxSxExLxxxD (SEQ ID NO:94), and/or GGxHRLxNYxS (SEQ IDNO:95). In addition, more specific consensus sequences can berepresented by aligning subsets of the 14 members of the ArabidopsisPYR/PYL proteins. Examples of such consensus sequences include, e.g.,

PYR1 to PYL12 (SEQ ID NO: 96) CxSxxxxxxxAPxxxxWxxxxxFxxPxxxKxFxxxC(SEQ ID NO: 97) GxxRxVxxxSxLPAxxSxExLxxxD (SEQ ID NO: 98) GGxHRLxNYxS(SEQ ID NO: 99) ESxxVDxPxGNxxxxTxxFxxxxxxxNLxxL PYR1-PYL6(SEQ ID NO: 100) HxxxxxxxxCxSxxxxxxxAPxxxxWxxxxxFxxPxxYKxFxxxC(SEQ ID NO: 101) VGRxVxVxSGLPAxxSxExLxxxDxxxxxxxFxxxGGxHRLxNYxSVT(SEQ ID NO: 102) VxESYxVDxPxGNxxxxTxxFxDxxxxxNLQxL PYL7-PYL10(SEQ ID NO: 103) HxHxxxxxQCxSxLVKxIxAPxHxVWSxVRRFDxPQKYKPFxSRCxVxGx(SEQ ID NO: 104)ExGxxREVxxKSGLPATxSTExLExLDDxEHILxIxIxGGDHRLKNYSSxxxxHxExIxGxxGTx(SEQ ID NO: 105) xxESFVVDVPxGNTKxxTCxFVExLIxCNLxSLAxxxERL PYL11-PYL13(SEQ ID NO: 106) CxSxxVxTIxAPLxLVWSILRxFDxPxxxxxFVKxCxxxSGxGG(SEQ ID NO: 107) GSVRxVTxVSxxPAxFSxERLxELDDESHVMxxSIIGGxHRLVNYxSKTAccordingly, in some embodiments, the PYR/PYL polypeptides of theinvention comprise one or more of the above-described consensussequences or conservative variants thereof.

Those of skill in the art will recognize that the variable positionswithin the above consensus sequences can be selected based on what aminoacids occur at their corresponding positions in specific PYR1polypeptides (e.g., as occur in any of SEQ ID NOs:2-90) or alternativelycan be conservative substitutions thereof. In some embodiments, thePYR/PYL polypeptides of the invention are substantially identical to(e.g., at least 70%, 75%, 80%, 85%, 90%, 95% identical to) any of SEQ IDNO:2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20,21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38,39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56,57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74,75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 108,109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122,123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, or137.

The present invention provides for use of the above proteins and/ornucleic acid sequences, encoding such polypeptides, in the methods andcompositions (e.g., expression cassettes, plants, etc.) of the presentinvention. The isolation of a polynucleotide sequence encoding a plantPYR/PYL (e.g., from plants where PYR/PYL sequences have not yet beenidentified) may be accomplished by a number of techniques. For instance,oligonucleotide probes based on the PYR/PYL coding sequences disclosed(e.g., as listed in the SEQUENCE LISTING) here can be used to identifythe desired PYR/PYL gene in a cDNA or genomic DNA library. To constructgenomic libraries, large segments of genomic DNA are generated by randomfragmentation, e.g., using restriction endonucleases, and are ligatedwith vector DNA to form concatemers that can be packaged into theappropriate vector. To prepare a cDNA library, mRNA is isolated from thedesired tissue, such as a leaf from a particular plant species, and acDNA library containing the gene transcript of interest is prepared fromthe mRNA. Alternatively, cDNA may be prepared from mRNA extracted fromother tissues in which PYR/PYL gene is expressed.

The cDNA or genomic library can then be screened using a probe basedupon the sequence of a PYR/PYL gene disclosed here. Probes may be usedto hybridize with genomic DNA or cDNA sequences to isolate homologousgenes in the same or different plant species. Alternatively, antibodiesraised against a polypeptide can be used to screen an mRNA expressionlibrary.

Alternatively, the nucleic acids encoding PYR/PYL can be amplified fromnucleic acid samples using amplification techniques. For instance,polymerase chain reaction (PCR) technology can be used to amplify thecoding sequences of PYR/PYL directly from genomic DNA, from cDNA, fromgenomic libraries or cDNA libraries. PCR and other in vitroamplification methods may also be useful, for example, to clonepolynucleotide sequences encoding PYR/PYL to be expressed, to makenucleic acids to use as probes for detecting the presence of the desiredmRNA in samples, for nucleic acid sequencing, or for other purposes. Fora general overview of PCR see PCR Protocols: A Guide to Methods andApplications. (Innis, M, Gelfand, D., Sninsky, J. and White, T., eds.),Academic Press, San Diego (1990). Appropriate primers and probes foridentifying sequences from plant tissues are generated from comparisonsof the sequences provided here with other related genes.

In some embodiments, the partial or entire genome of a number of plantshas been sequenced and open reading frames identified. By a BLASTsearch, one can identify the coding sequence for PYR/PYL in variousplants.

Variants from naturally-occurring PYR/PYL polypeptides (or nucleic acidsencoding such polypeptides) are contemplated by the term PYR/PYLpolypeptide. Variants include, e.g., fusion proteins, deletions ormutations that retain activity.

In some embodiments, the PYR/PYL polypeptide is activated (e.g., asmeasured in a two-hybrid assay with PP2C or other receptor assays) inthe presence of ABA (or ABA agonist) but is not significantly active inthe absence of ABA or agonist. Alternatively, in some embodiments, thePYR/PYL polypeptides of the invention are constitutively active, i.e.,are active in the absence of ABA or an ABA agonist. As described in theExamples, the inventors have found that the mutations H60P, M158T,M158I, M158S, or M158V in Arabidopsis PYR1 changes the protein to aconstitutively active protein. As both of these positions (H60 and M158)are present on the dimer interface of the PYR/PYL protein, it isbelieved that other constitutive mutants can be generated by introducingamino acid changes at other dimer interface positions (e.g., F61, K63,184, S85, L87, P88, A89, S152, D155, T156, F159, T162, L166, and/orK170). While the positions above are made with reference to theArabidopsis PYR1 protein, it is intended that the corresponding positionin other PYR/PYL polypeptides are also included in the abovedescription. The corresponding position in another PYR/PYL polypeptidecan be readily determined using standard alignment software such asBLAST. While specific amino acid changes are described above, theinvention is intended to encompass mutations to other amino acids asidethose specifically described above. In some embodiments, for example,conservative amino acids can be included in place of the mutations setforth above.

Interestingly, the inventors have observed that some naturally occurringPYR/PYL proteins naturally have a P at the position that corresponds toH60. For example, Arabidopsis PYL9 has a P at this position. Theinventors have found that PYL9 is constitutively active. In someembodiments, a constitutively active PYR/PYL protein is converted to aprotein activated by ABA or an ABA agonist by changing a proline atposition “H60” (with reference to the position in Arabidopsis PYR1) to aHistidine or other non-proline amino acid.

Accordingly, the present invention provides for PYR/PYL polypeptidesthat are constitutively active and having a mutation as described above.In some embodiments, the constitutive polypeptides will comprise one ormore of the above-described consensus sequences and/or will besubstantially identical to one of SEQ ID NOs:2-90.

VI. Use of PYR/PYL Nucleic Acids and Polypeptides of the Invention

The invention provides methods of modulating ABA sensitivity in a plantby altering PYR/PYL expression or activity, for example, by introducinginto a plant a recombinant expression cassette comprising a regulatoryelement (e.g., a promoter) operably linked to a PYR/PYL polynucleotide,i.e., a nucleic acid encoding PYR/PYL or a sequence comprising a portionof the sequence of a PYR/PYL mRNA or complement thereof.

In some embodiments, the methods of the invention comprise increasingand/or ectopically expressing one or more PYR/PYL polynucleotideencoding a PYR/PYL polypeptide in a plant. Such embodiments are usefulfor increasing ABA sensitivity of a plant, and resulting in, forexample, improved stress (e.g., drought) tolerance and/or delayed seedgermination (to avoid pre-mature germination, for example as can occurin humid environments or due to other exposure to moisture). For stresstolerance, promoters can be selected that are generally constitutive andare expressed in most plant tissues, or can be leaf or root specific. Toaffect seed germination, promoters are generally used that result inexpression in seed or, in some embodiments, floral organs or embryos.

In some embodiments, the methods of the invention comprise decreasingendogenous PYR/PYL expression in plant, thereby decreasing ABAsensitivity in the plant. Such methods can involve, for example,mutagenesis (e.g., chemical, radiation, transposon or other mutagenesis)of PYR/PYL sequences in a plant to reduce PYR/PYL expression oractivity, or introduction of a polynucleotide substantially identical toat least a portion of a PYR/PYL cDNA sequence or a complement thereof(e.g., an “RNAi construct”) to reduce PYR/PYL expression. Decreased (orincreased) PYR/PYL expression can be used to control the development ofabscission zones in leaf petioles and thereby control leaf loss, i.e.,delay leaf loss if expression is decreased and speed leaf loss ifexpression is increased in abscission zones in a leaf.

A. Increasing PYR/PYL Expression or Activity

Isolated sequences prepared as described herein can also be used toprepare expression cassettes that enhance or increase PYR/PYL geneexpression. Where overexpression of a gene is desired, the desired gene(or at least the polynucleotide encoding a PYR/PYL polypeptide) from thesame species or a different species (or substantially identical to thegene or polynucleotide encoding a PYR/PYL polypeptide from anotherspecies) may be used. In some embodiments, to decrease potential sensesuppression effects, a polynucleotide encoding a PYR/PYL polypeptidefrom a different species (or substantially identical to the gene orpolynucleotide encoding a PYR/PYL polypeptide from another species) maybe used.

Any of a number of means well known in the art can be used to increasePYR/PYL activity in plants. Any organ or plant part can be targeted,such as shoot vegetative organs/structures (e.g. leaves, stems andtubers), roots, flowers and floral organs/structures (e.g. bracts,sepals, petals, stamens, carpels, anthers and ovules), seed (includingembryo, endosperm, and seed coat), fruit, abscission zone, etc.Alternatively, one or several PYR/PYL genes can be expressedconstitutively (e.g., using the CaMV 35S promoter or other constitutivepromoter).

One of skill will recognize that the polypeptides encoded by the genesof the invention, like other proteins, have different domains whichperform different functions. Thus, the overexpressed or ectopicallyexpressed polynucleotide sequences need not be full length, so long asthe desired functional domain of the protein is expressed.Alternatively, or in addition, active PYR/PYL proteins can be expressedas fusions, without necessarily significantly altering PYR/PYL activity.Examples of fusion partners include, but are not limited to, poly-His orother tag sequences.

B. Decreasing PYR/PYL Expression or Activity

A number of methods can be used to inhibit gene expression in plants. Avariety of methods to inhibit gene expression are known and can be usedto inhibit expression of one of more PYR/PYL genes. See, e.g., U.S. Pat.Nos. 5,759,829; 5,107,065; 5,231,020; 5,283,184; 6,506,559; 6,573,099,6,326,193; 7,109,393. For instance, antisense technology can beconveniently used. To accomplish this, a nucleic acid segment from thedesired gene is cloned and operably linked to a promoter such that theantisense strand of RNA will be transcribed. The expression cassette isthen transformed into plants and the antisense strand of RNA isproduced. In plant cells, it has been suggested that antisense RNAinhibits gene expression by preventing the accumulation of mRNA whichencodes the enzyme of interest, see, e.g., Sheehy et al., Proc. Nat.Acad. Sci. USA, 85:8805-8809 (1988); Pnueli et al., The Plant Cell6:175-186 (1994); and Hiatt et al., U.S. Pat. No. 4,801,340.

The antisense nucleic acid sequence transformed into plants will besubstantially identical to at least a portion of the endogenous gene orgenes to be repressed. The sequence, however, does not have to beperfectly identical to inhibit expression. Thus, an antisense or sensenucleic acid molecule encoding only a portion of PYR/PYL polypeptide, ora portion of the PYR/PYL cDNA, can be useful for producing a plant inwhich PYR/PYL expression is suppressed. The vectors of the presentinvention can be designed such that the inhibitory effect applies toother proteins within a family of genes exhibiting homology orsubstantial homology to the target gene. In some embodiments, it may bedesirable to inhibit the expression of more than one PYR/PYL polypeptideat the same time using one or more antisense or sense or other siRNAnucleic acid molecules.

For antisense suppression, the introduced sequence also need not be fulllength relative to either the primary transcription product or fullyprocessed mRNA. Generally, higher homology can be used to compensate forthe use of a shorter sequence. Furthermore, the introduced sequence neednot have the same intron or exon pattern, and homology of non-codingsegments may be equally effective. For example, a sequence of betweenabout 30 or 40 nucleotides can be used, and in some embodiments, aboutfull length nucleotides should be used, though a sequence of at leastabout 20, 50 100, 200, or 500 nucleotides can be used.

Catalytic RNA molecules or ribozymes can also be used to inhibitexpression of PYR/PYL genes. It is possible to design ribozymes thatspecifically pair with virtually any target RNA and cleave thephosphodiester backbone at a specific location, thereby functionallyinactivating the target RNA. In carrying out this cleavage, the ribozymeis not itself altered, and is thus capable of recycling and cleavingother molecules, making it a true enzyme. The inclusion of ribozymesequences within antisense RNAs confers RNA-cleaving activity upon them,thereby increasing the activity of the constructs.

A number of classes of ribozymes have been identified. One class ofribozymes is derived from a number of small circular RNAs that arecapable of self-cleavage and replication in plants. The RNAs replicateeither alone (viroid RNAs) or with a helper virus (satellite RNAs).Examples include RNAs from avocado sunblotch viroid and the satelliteRNAs from tobacco ringspot virus, lucerne transient streak virus, velvettobacco mottle virus, solanum nodiflorum mottle virus and subterraneanclover mottle virus. The design and use of target RNA-specific ribozymesis described in Haseloff et al. Nature, 334:585-591 (1988).

Another method of suppression is sense suppression (also known asco-suppression). Introduction of expression cassettes in which a nucleicacid is configured in the sense orientation with respect to the promoterhas been shown to be an effective means by which to block thetranscription of target genes. For an example of the use of this methodto modulate expression of endogenous genes see, Napoli et al., The PlantCell 2:279-289 (1990); Flavell, Proc. Natl. Acad. Sci., USA 91:3490-3496(1994); Kooter and Mol, Current Opin. Biol. 4:166-171 (1993); and U.S.Pat. Nos. 5,034,323, 5,231,020, and 5,283,184.

Generally, where inhibition of expression is desired, some transcriptionof the introduced sequence occurs. The effect may occur where theintroduced sequence contains no coding sequence per se, but only intronor untranslated sequences homologous to sequences present in the primarytranscript of the endogenous sequence. The introduced sequence generallywill be substantially identical to the endogenous sequence intended tobe repressed. This minimal identity will typically be greater than about65%, but a higher identity can exert a more effective repression ofexpression of the endogenous sequences. In some embodiments, sequenceswith substantially greater identity are used, e.g., at least about 80,at least about 95%, or 100% identity are used. As with antisenseregulation, the effect can be designed and tested to apply to any otherproteins within a similar family of genes exhibiting homology orsubstantial homology.

For sense suppression, the introduced sequence in the expressioncassette, needing less than absolute identity, also need not be fulllength, relative to either the primary transcription product or fullyprocessed mRNA. This may be preferred to avoid concurrent production ofsome plants that are overexpressers. A higher identity in a shorter thanfull length sequence compensates for a longer, less identical sequence.Furthermore, the introduced sequence need not have the same intron orexon pattern, and identity of non-coding segments will be equallyeffective. In some embodiments, a sequence of the size ranges notedabove for antisense regulation is used, i.e., 30-40, or at least about20, 50, 100, 200, 500 or more nucleotides.

Endogenous gene expression may also be suppressed by means of RNAinterference (RNAi) (and indeed co-suppression can be considered a typeof RNAi), which uses a double-stranded RNA having a sequence identicalor similar to the sequence of the target gene. RNAi is the phenomenon inwhich when a double-stranded RNA having a sequence identical or similarto that of the target gene is introduced into a cell, the expressions ofboth the inserted exogenous gene and target endogenous gene aresuppressed. The double-stranded RNA may be formed from two separatecomplementary RNAs or may be a single RNA with internally complementarysequences that form a double-stranded RNA. Although complete details ofthe mechanism of RNAi are still unknown, it is considered that theintroduced double-stranded RNA is initially cleaved into smallfragments, which then serve as indexes of the target gene in somemanner, thereby degrading the target gene. RNAi is known to be alsoeffective in plants (see, e.g., Chuang, C. F. & Meyerowitz, E. M., Proc.Natl. Acad. Sci. USA 97: 4985 (2000); Waterhouse et al., Proc. Natl.Acad. Sci. USA 95:13959-13964 (1998); Tabara et al. Science 282:430-431(1998); Matthew, Comp Funct Genom 5: 240-244 (2004); Lu, et al., NucleicAcids Research 32(21):e171 (2004)). For example, to achieve suppressionof the expression of a DNA encoding a protein using RNAi, adouble-stranded RNA having the sequence of a DNA encoding the protein,or a substantially similar sequence thereof (including those engineerednot to translate the protein) or fragment thereof, is introduced into aplant of interest. The resulting plants may then be screened for aphenotype associated with the target protein and/or by monitoringsteady-state RNA levels for transcripts encoding the protein. Althoughthe genes used for RNAi need not be completely identical to the targetgene, they may be at least 70%, 80%, 90%, 95% or more identical to thetarget (e.g., PYR/PYL) gene sequence. See, e.g., U.S. Patent PublicationNo. 2004/0029283. The constructs encoding an RNA molecule with astem-loop structure that is unrelated to the target gene and that ispositioned distally to a sequence specific for the gene of interest mayalso be used to inhibit target gene expression. See, e.g., U.S. PatentPublication No. 2003/0221211.

The RNAi polynucleotides can encompass the full-length target RNA or maycorrespond to a fragment of the target RNA. In some cases, the fragmentwill have fewer than 100, 200, 300, 400, 500 600, 700, 800, 900 or 1,000nucleotides corresponding to the target sequence. In addition, in someembodiments, these fragments are at least, e.g., 50, 100, 150, 200, ormore nucleotides in length. In some cases, fragments for use in RNAiwill be at least substantially similar to regions of a target proteinthat do not occur in other proteins in the organism or may be selectedto have as little similarity to other organism transcripts as possible,e.g., selected by comparison to sequences in analyzingpublicly-available sequence databases.

Expression vectors that continually express siRNA in transiently- andstably-transfected have been engineered to express small hairpin RNAs,which get processed in vivo into siRNAs molecules capable of carryingout gene-specific silencing (Brummelkamp et al., Science 296:550-553(2002), and Paddison, et al., Genes & Dev. 16:948-958 (2002)).Post-transcriptional gene silencing by double-stranded RNA is discussedin further detail by Hammond et al. Nature Rev Gen 2: 110-119 (2001),Fire et al. Nature 391: 806-811 (1998) and Timmons and Fire Nature 395:854 (1998).

One of skill in the art will recognize that using technology based onspecific nucleotide sequences (e.g., antisense or sense suppressiontechnology), families of homologous genes can be suppressed with asingle sense or antisense transcript. For instance, if a sense orantisense transcript is designed to have a sequence that is conservedamong a family of genes, then multiple members of a gene family can besuppressed. Conversely, if the goal is to only suppress one member of ahomologous gene family, then the sense or antisense transcript should betargeted to sequences with the most variance between family members.

Another means of inhibiting PYR/PYL function in a plant is by creationof dominant negative mutations. In this approach, non-functional, mutantPYR/PYL polypeptides, which retain the ability to interact withwild-type subunits are introduced into a plant. A dominant negativeconstruct also can be used to suppress PYR/PYL expression in a plant. Adominant negative construct useful in the invention generally contains aportion of the complete PYR/PYL coding sequence sufficient, for example,for DNA-binding or for a protein-protein interaction such as ahomodimeric or heterodimeric protein-protein interaction but lacking thetranscriptional activity of the wild type protein.

VII. Recombinant Expression Vectors

Once the coding or cDNA sequence for PYR/PYL is obtained, it can also beused to prepare an expression cassette for expressing the PYR/PYLprotein in a transgenic plant, directed by a heterologous promoter.Increased expression of PYR/PYL polynucleotide is useful, for example,to produce plants with enhanced drought-resistance. Alternatively, asdescribed above, expression vectors can also be used to express PYR/PYLpolynucleotides and variants thereof that inhibit endogenous PYR/PYLexpression.

Any of a number of means well known in the art can be used to increaseor decrease PYR/PYL activity or expression in plants. Any organ can betargeted, such as shoot vegetative organs/structures (e.g. leaves, stemsand tubers), roots, flowers and floral organs/structures (e.g. bracts,sepals, petals, stamens, carpels, anthers and ovules), seed (includingembryo, endosperm, and seed coat) and fruit. Alternatively, the PYR/PYLgene can be expressed constitutively (e.g., using the CaMV 35Spromoter).

To use PYR/PYL coding or cDNA sequences in the above techniques,recombinant DNA vectors suitable for transformation of plant cells areprepared. Techniques for transforming a wide variety of higher plantspecies are well known and described in the technical and scientificliterature. See, e.g., Weising et al. Ann. Rev. Genet. 22:421-477(1988). A DNA sequence coding for the PYR/PYL polypeptide preferablywill be combined with transcriptional and translational initiationregulatory sequences which will direct the transcription of the sequencefrom the gene in the intended tissues of the transformed plant.

For example, a plant promoter fragment may be employed to directexpression of the PYR/PYL gene in all tissues of a regenerated plant.Such promoters are referred to herein as “constitutive” promoters andare active under most environmental conditions and states of developmentor cell differentiation. Examples of constitutive promoters include thecauliflower mosaic virus (CaMV) 35S transcription initiation region, the1′- or 2′-promoter derived from T-DNA of Agrobacterium tumafaciens, andother transcription initiation regions from various plant genes known tothose of skill.

Alternatively, the plant promoter may direct expression of the PYR/PYLprotein in a specific tissue (tissue-specific promoters) or may beotherwise under more precise environmental control (induciblepromoters). Examples of tissue-specific promoters under developmentalcontrol include promoters that initiate transcription only in certaintissues, such as leaves or guard cells (including but not limited tothose described in WO/2005/085449; U.S. Pat. No. 6,653,535; Li et al.,Sci China C Life Sci. 2005 April; 48(2):181-6; Husebye, et al., PlantPhysiol, April 2002, Vol. 128, pp. 1180-1188; and Plesch, et al., Gene,Volume 249, Number 1, 16 May 2000, pp. 83-89(7)). Examples ofenvironmental conditions that may affect transcription by induciblepromoters include anaerobic conditions, elevated temperature, or thepresence of light.

If proper protein expression is desired, a polyadenylation region at the3′-end of the coding region should be included. The polyadenylationregion can be derived from the natural gene, from a variety of otherplant genes, or from T-DNA.

The vector comprising the sequences (e.g., promoters or PYR/PYL codingregions) will typically comprise a marker gene that confers a selectablephenotype on plant cells. For example, the marker may encode biocideresistance, particularly antibiotic resistance, such as resistance tokanamycin, G418, bleomycin, hygromycin, or herbicide resistance, such asresistance to chlorosluforon or Basta.

In some embodiments, the PYR/PYL nucleic acid sequence is expressedrecombinantly in plant cells to enhance and increase levels of totalPYR/PYL polypeptide. A variety of different expression constructs, suchas expression cassettes and vectors suitable for transformation of plantcells can be prepared. Techniques for transforming a wide variety ofhigher plant species are well known and described in the technical andscientific literature. See, e.g., Weising et al. Ann. Rev. Genet.22:421-477 (1988). A DNA sequence coding for a PYR/PYL protein can becombined with cis-acting (promoter) and trans-acting (enhancer)transcriptional regulatory sequences to direct the timing, tissue typeand levels of transcription in the intended tissues of the transformedplant. Translational control elements can also be used.

The invention provides a PYR/PYL nucleic acid operably linked to apromoter which, in some embodiments, is capable of driving thetranscription of the PYR/PYL coding sequence in plants. The promoter canbe, e.g., derived from plant or viral sources. The promoter can be,e.g., constitutively active, inducible, or tissue specific. Inconstruction of recombinant expression cassettes, vectors, transgenics,of the invention, a different promoters can be chosen and employed todifferentially direct gene expression, e.g., in some or all tissues of aplant or animal.

A. Constitutive Promoters

A promoter fragment can be employed to direct expression of a PYR/PYLnucleic acid in all transformed cells or tissues, e.g., as those of aregenerated plant. The term “constitutive regulatory element” means aregulatory element that confers a level of expression upon anoperatively linked nucleic molecule that is relatively independent ofthe cell or tissue type in which the constitutive regulatory element isexpressed. A constitutive regulatory element that is expressed in aplant generally is widely expressed in a large number of cell and tissuetypes. Promoters that drive expression continuously under physiologicalconditions are referred to as “constitutive” promoters and are activeunder most environmental conditions and states of development or celldifferentiation.

A variety of constitutive regulatory elements useful for ectopicexpression in a transgenic plant are well known in the art. Thecauliflower mosaic virus 35S (CaMV 35S) promoter, for example, is awell-characterized constitutive regulatory element that produces a highlevel of expression in all plant tissues (Odell et al., Nature313:810-812 (1985)). The CaMV 35S promoter can be particularly usefuldue to its activity in numerous diverse plant species (Benfey and Chua,Science 250:959-966 (1990); Futterer et al., Physiol. Plant 79:154(1990); Odell et al., supra, 1985). A tandem 35S promoter, in which theintrinsic promoter element has been duplicated, confers higherexpression levels in comparison to the unmodified 35S promoter (Kay etal., Science 236:1299 (1987)). Other useful constitutive regulatoryelements include, for example, the cauliflower mosaic virus 19Spromoter; the Figwort mosaic virus promoter; and the nopaline synthase(nos) gene promoter (Singer et al., Plant Mol. Biol. 14:433 (1990); An,Plant Physiol. 81:86 (1986)).

Additional constitutive regulatory elements including those forefficient expression in monocots also are known in the art, for example,the pEmu promoter and promoters based on the rice Actin-1 5′ region(Last et al., Theor. Appl. Genet. 81:581 (1991); Mcelroy et al., Mol.Gen. Genet. 231:150 (1991); Mcelroy et al., Plant Cell 2:163 (1990)).Chimeric regulatory elements, which combine elements from differentgenes, also can be useful for ectopically expressing a nucleic acidmolecule encoding a PYR/PYL protein (Comai et al., Plant Mol. Biol.15:373 (1990)).

Other examples of constitutive promoters include the 1′- or 2′-promoterderived from T-DNA of Agrobacterium tumafaciens (see, e.g., Mengiste(1997) supra; O'Grady (1995) Plant Mol. Biol. 29:99-108); actinpromoters, such as the Arabidopsis actin gene promoter (see, e.g., Huang(1997) Plant Mol. Biol. 1997 33:125-139); alcohol dehydrogenase (Adh)gene promoters (see, e.g., Millar (1996) Plant Mol. Biol. 31:897-904);ACT11 from Arabidopsis (Huang et al. Plant Mol. Biol. 33:125-139(1996)), Cat3 from Arabidopsis (GenBank No. U43147, Zhong et al., Mol.Gen. Genet. 251:196-203 (1996)), the gene encoding stearoyl-acyl carrierprotein desaturase from Brassica napus (Genbank No. X74782, Solocombe etal. Plant Physiol. 104:1167-1176 (1994)), GPc1 from maize (GenBank No.X15596, Martinez et al. J. Mol. Biol. 208:551-565 (1989)), Gpc2 frommaize (GenBank No. U45855, Manjunath et al., Plant Mol. Biol. 33:97-112(1997)), other transcription initiation regions from various plant genesknown to those of skill. See also Holtorf Plant Mol. Biol. 29:637-646(1995).

B. Inducible Promoters

Alternatively, a plant promoter may direct expression of the PYR/PYLgene under the influence of changing environmental conditions ordevelopmental conditions. Examples of environmental conditions that mayeffect transcription by inducible promoters include anaerobicconditions, elevated temperature, drought, or the presence of light.Such promoters are referred to herein as “inducible” promoters. Forexample, the invention can incorporate drought-specific promoter such asthe drought-inducible promoter of maize (Busk (1997) supra); oralternatively the cold, drought, and high salt inducible promoter frompotato (Kirch (1997) Plant Mol. Biol. 33:897-909).

Alternatively, plant promoters which are inducible upon exposure toplant hormones, such as auxins, are used to express the PYR/PYL gene.For example, the invention can use the auxin-response elements E1promoter fragment (AuxREs) in the soybean (Glycine max L.) (Liu (1997)Plant Physiol. 115:397-407); the auxin-responsive Arabidopsis GST6promoter (also responsive to salicylic acid and hydrogen peroxide) (Chen(1996) Plant J. 10: 955-966); the auxin-inducible parC promoter fromtobacco (Sakai (1996) 37:906-913); a plant biotin response element(Streit (1997) Mol. Plant. Microbe Interact. 10:933-937); and, thepromoter responsive to the stress hormone abscisic acid (Sheen (1996)Science 274:1900-1902).

Plant promoters inducible upon exposure to chemicals reagents that maybe applied to the plant, such as herbicides or antibiotics, are alsouseful for expressing the PYR/PYL gene. For example, the maize In2-2promoter, activated by benzenesulfonamide herbicide safeners, can beused (De Veylder (1997) Plant Cell Physiol. 38:568-577); application ofdifferent herbicide safeners induces distinct gene expression patterns,including expression in the root, hydathodes, and the shoot apicalmeristem. A PYR/PYL coding sequence can also be under the control of,e.g., a tetracycline-inducible promoter, e.g., as described withtransgenic tobacco plants containing the Avena sativa L. (oat) argininedecarboxylase gene (Masgrau (1997) Plant J. 11:465-473); or, a salicylicacid-responsive element (Stange (1997) Plant J. 11:1315-1324; Uknes etal., Plant Cell 5:159-169 (1993); Bi et al., Plant J. 8:235-245 (1995)).

Examples of useful inducible regulatory elements includecopper-inducible regulatory elements (Mett et al., Proc. Natl. Acad.Sci. USA 90:4567-4571 (1993); Furst et al., Cell 55:705-717 (1988));tetracycline and chlor-tetracycline-inducible regulatory elements (Gatzet al., Plant J. 2:397-404 (1992); Röder et al., Mol. Gen. Genet.243:32-38 (1994); Gatz, Meth. Cell Biol. 50:411-424 (1995)); ecdysoneinducible regulatory elements (Christopherson et al., Proc. Natl. Acad.Sci. USA 89:6314-6318 (1992); Kreutzweiser et al., Ecotoxicol. Environ.Safety 28:14-24 (1994)); heat shock inducible regulatory elements(Takahashi et al., Plant Physiol. 99:383-390 (1992); Yabe et al., PlantCell Physiol. 35:1207-1219 (1994); Ueda et al., Mol. Gen. Genet.250:533-539 (1996)); and lac operon elements, which are used incombination with a constitutively expressed lac repressor to confer, forexample, IPTG-inducible expression (Wilde et al., EMBO J. 11:1251-1259(1992)). An inducible regulatory element useful in the transgenic plantsof the invention also can be, for example, a nitrate-inducible promoterderived from the spinach nitrite reductase gene (Back et al., Plant Mol.Biol. 17:9 (1991)) or a light-inducible promoter, such as thatassociated with the small subunit of RuBP carboxylase or the LHCP genefamilies (Feinbaum et al., Mol. Gen. Genet. 226:449 (1991); Lam andChua, Science 248:471 (1990)).

C. Tissue-Specific Promoters

Alternatively, the plant promoter may direct expression of the PYR/PYLgene in a specific tissue (tissue-specific promoters). Tissue specificpromoters are transcriptional control elements that are only active inparticular cells or tissues at specific times during plant development,such as in vegetative tissues or reproductive tissues.

Examples of tissue-specific promoters under developmental controlinclude promoters that initiate transcription only (or primarily only)in certain tissues, such as vegetative tissues, e.g., roots or leaves,or reproductive tissues, such as fruit, ovules, seeds, pollen, pistols,flowers, or any embryonic tissue, or epidermis or mesophyll.Reproductive tissue-specific promoters may be, e.g., ovule-specific,embryo-specific, endosperm-specific, integument-specific, seed and seedcoat-specific, pollen-specific, petal-specific, sepal-specific, or somecombination thereof. In some embodiments, the promoter is cell-typespecific, e.g., guard cell-specific.

Other tissue-specific promoters include seed promoters. Suitableseed-specific promoters are derived from the following genes: MAC1 frommaize (Sheridan (1996) Genetics 142:1009-1020); Cat3 from maize (GenBankNo. L05934, Abler (1993) Plant Mol. Biol. 22:10131-1038); vivparous-1from Arabidopsis (Genbank No. U93215); atmyc 1 from Arabidopsis (Urao(1996) Plant Mol. Biol. 32:571-57; Conceicao (1994) Plant 5:493-505);napA from Brassica napus (GenBank No. J02798, Josefsson (1987) JBL26:12196-1301); and the napin gene family from Brassica napus (Sjodahl(1995) Planta 197:264-271).

A variety of promoters specifically active in vegetative tissues, suchas leaves, stems, roots and tubers, can also be used to expresspolynucleotides encoding PYR/PYL polypeptides (or RNAi or antisense orsense constructs). For example, promoters controlling patatin, the majorstorage protein of the potato tuber, can be used, see, e.g., Kim (1994)Plant Mol. Biol. 26:603-615; Martin (1997) Plant J. 11:53-62. The ORF13promoter from Agrobacterium rhizogenes that exhibits high activity inroots can also be used (Hansen (1997) Mol. Gen. Genet. 254:337-343.Other useful vegetative tissue-specific promoters include: the tarinpromoter of the gene encoding a globulin from a major taro (Colocasiaesculenta L. Schott) corm protein family, tarin (Bezerra (1995) PlantMol. Biol. 28:137-144); the curculin promoter active during taro cormdevelopment (de Castro (1992) Plant Cell 4:1549-1559) and the promoterfor the tobacco root-specific gene TobRB7, whose expression is localizedto root meristem and immature central cylinder regions (Yamamoto (1991)Plant Cell 3:371-382).

Leaf-specific promoters, such as the ribulose biphosphate carboxylase(RBCS) promoters can be used. For example, the tomato RBCS1, RBCS2 andRBCS3A genes are expressed in leaves and light-grown seedlings, onlyRBCS1 and RBCS2 are expressed in developing tomato fruits (Meier (1997)FEBS Lett. 415:91-95). A ribulose bisphosphate carboxylase promotersexpressed almost exclusively in mesophyll cells in leaf blades and leafsheaths at high levels, described by Matsuoka (1994) Plant J. 6:311-319,can be used. Another leaf-specific promoter is the light harvestingchlorophyll a/b binding protein gene promoter, see, e.g., Shiina (1997)Plant Physiol. 115:477-483; Casal (1998) Plant Physiol. 116:1533-1538.The Arabidopsis thaliana myb-related gene promoter (Atmyb5) described byLi (1996) FEBS Lett. 379:117-121, is leaf-specific. The Atmyb5 promoteris expressed in developing leaf trichomes, stipules, and epidermal cellson the margins of young rosette and cauline leaves, and in immatureseeds. Atmyb5 mRNA appears between fertilization and the 16 cell stageof embryo development and persists beyond the heart stage. A leafpromoter identified in maize by Busk (1997) Plant J. 11:1285-1295, canalso be used.

Another class of useful vegetative tissue-specific promoters aremeristematic (root tip and shoot apex) promoters. For example, the“SHOOTMERISTEMLESS” and “SCARECROW” promoters, which are active in thedeveloping shoot or root apical meristems, described by Di Laurenzio(1996) Cell 86:423-433; and, Long (1996) Nature 379:66-69; can be used.Another useful promoter is that which controls the expression of3-hydroxy-3-methylglutaryl coenzyme A reductase HMG2 gene, whoseexpression is restricted to meristematic and floral (secretory zone ofthe stigma, mature pollen grains, gynoecium vascular tissue, andfertilized ovules) tissues (see, e.g., Enjuto (1995) Plant Cell.7:517-527). Also useful are kn1-related genes from maize and otherspecies which show meristem-specific expression, see, e.g., Granger(1996) Plant Mol. Biol. 31:373-378; Kerstetter (1994) Plant Cell6:1877-1887; Hake (1995) Philos. Trans. R. Soc. Lond. B. Biol. Sci.350:45-51. For example, the Arabidopsis thaliana KNAT1 promoter (see,e.g., Lincoln (1994) Plant Cell 6:1859-1876).

One of skill will recognize that a tissue-specific promoter may driveexpression of operably linked sequences in tissues other than the targettissue. Thus, as used herein a tissue-specific promoter is one thatdrives expression preferentially in the target tissue, but may also leadto some expression in other tissues as well.

In another embodiment, the PYR/PYL polynucleotide is expressed through atransposable element. This allows for constitutive, yet periodic andinfrequent expression of the constitutively active polypeptide. Theinvention also provides for use of tissue-specific promoters derivedfrom viruses including, e.g., the tobamovirus subgenomic promoter(Kumagai (1995) Proc. Natl. Acad. Sci. USA 92:1679-1683; the rice tungrobacilliform virus (RTBV), which replicates only in phloem cells ininfected rice plants, with its promoter which drives strongphloem-specific reporter gene expression; the cassaya vein mosaic virus(CVMV) promoter, with highest activity in vascular elements, in leafmesophyll cells, and in root tips (Verdaguer (1996) Plant Mol. Biol.31:1129-1139).

VIII. Production of Transgenic Plants

As detailed herein, the present invention provides for transgenic plantscomprising recombinant expression cassettes either for expressingPYR/PYL proteins in a plant or for inhibiting or reducing endogenousPYR/PYL expression. Thus, in some embodiments, a transgenic plant isgenerated that contains a complete or partial sequence of an endogenousPYR/PYL encoding polynucleotide, either for increasing or reducingPYR/PYL expression and activity. In some embodiments, a transgenic plantis generated that contains a complete or partial sequence of apolynucleotide that is substantially identical to an endogenous PYR/PYLencoding polynucleotide, either for increasing or reducing PYR/PYLexpression and activity. In some embodiments, a transgenic plant isgenerated that contains a complete or partial sequence of apolynucleotide that is from a species other than the species of thetransgenic plant. It should be recognized that transgenic plantsencompass the plant or plant cell in which the expression cassette isintroduced as well as progeny of such plants or plant cells that containthe expression cassette, including the progeny that have the expressioncassette stably integrated in a chromosome.

A recombinant expression vector comprising a PYR/PYL coding sequencedriven by a heterologous promoter may be introduced into the genome ofthe desired plant host by a variety of conventional techniques. Forexample, the DNA construct may be introduced directly into the genomicDNA of the plant cell using techniques such as electroporation andmicroinjection of plant cell protoplasts, or the DNA construct can beintroduced directly to plant tissue using ballistic methods, such as DNAparticle bombardment. Alternatively, the DNA construct may be combinedwith suitable T-DNA flanking regions and introduced into a conventionalAgrobacterium tumefaciens host vector. The virulence functions of theAgrobacterium tumefaciens host will direct the insertion of theconstruct and adjacent marker into the plant cell DNA when the cell isinfected by the bacteria. While transient expression of PYR/PYL isencompassed by the invention, generally expression of construction ofthe invention will be from insertion of expression cassettes into theplant genome, e.g., such that at least some plant offspring also containthe integrated expression cassette.

Microinjection techniques are also useful for this purpose. Thesetechniques are well known in the art and thoroughly described in theliterature. The introduction of DNA constructs using polyethylene glycolprecipitation is described in Paszkowski et al. EMBO J. 3:2717-2722(1984). Electroporation techniques are described in Fromm et al. Proc.Natl. Acad. Sci. USA 82:5824 (1985). Ballistic transformation techniquesare described in Klein et al. Nature 327:70-73 (1987).

Agrobacterium tumefaciens-mediated transformation techniques, includingdisarming and use of binary vectors, are well described in thescientific literature. See, for example, Horsch et al. Science233:496-498 (1984), and Fraley et al. Proc. Natl. Acad. Sci. USA 80:4803(1983).

Transformed plant cells derived by any of the above transformationtechniques can be cultured to regenerate a whole plant that possessesthe transformed genotype and thus the desired phenotype such as enhanceddrought-resistance. Such regeneration techniques rely on manipulation ofcertain phytohormones in a tissue culture growth medium, typicallyrelying on a biocide and/or herbicide marker which has been introducedtogether with the desired nucleotide sequences. Plant regeneration fromcultured protoplasts is described in Evans et al., Protoplasts Isolationand Culture, Handbook of Plant Cell Culture, pp. 124-176, MacMillilanPublishing Company, New York, 1983; and Binding, Regeneration of Plants,Plant Protoplasts, pp. 21-73, CRC Press, Boca Raton, 1985. Regenerationcan also be obtained from plant callus, explants, organs, or partsthereof. Such regeneration techniques are described generally in Klee etal. Ann. Rev. of Plant Phys. 38:467-486 (1987).

One of skill will recognize that after the expression cassette is stablyincorporated in transgenic plants and confirmed to be operable, it canbe introduced into other plants by sexual crossing. Any of a number ofstandard breeding techniques can be used, depending upon the species tobe crossed.

The expression cassettes of the invention can be used to confer droughtresistance on essentially any plant. Thus, the invention has use over abroad range of plants, including species from the genera Asparagus,Atropa, Avena, Brassica, Citrus, Citrullus, Capsicum, Cucumis,Cucurbita, Daucus, Fragaria, Glycine, Gossypium, Helianthus,Heterocallis, Hordeum, Hyoscyamus, Lactuca, Linum, Lolium, Lycopersicon,Malus, Manihot, Majorana, Medicago, Nicotiana, Oryza, Panieum,Pannesetum, Persea, Pisum, Pyrus, Prunus, Raphanus, Secale, Senecio,Sinapis, Solanum, Sorghum, Trigonella, Triticum, Vitis, Vigna, and, Zea.In some embodiments, the plant is selected from the group consisting ofrice, maize, wheat, soybeans, cotton, canola, turfgrass, and alfalfa. Insome embodiments, the plant is an ornamental plant. In some embodiment,the plant is a vegetable- or fruit-producing plant.

Those of skill will recognize that a number of plant species can be usedas models to predict the phenotypic effects of transgene expression inother plants. For example, it is well recognized that both tobacco(Nicotiana) and Arabidopsis plants are useful models of transgeneexpression, particularly in other dicots.

The plants of the invention have either enhanced or reduced abscisicacid sensitivity compared to plants are otherwise identical except forexpression of PYR/PYL. Abscisic acid sensitivity can be monitored byobserving or measuring any phenotype mediated by ABA. Those of skill inthe art will recognize that ABA is a well-studied plant hormone and thatABA mediates many changes in characteristics, any of which can bemonitored to determined whether ABA sensitivity has been modulated. Insome embodiments, modulated ABA sensitivity is manifested by alteredtiming of seed germination or altered stress (e.g., drought) tolerance.

Drought resistance can assayed according to any of a number ofwell-known techniques. For example, plants can be grown under conditionsin which less than optimum water is provided to the plant. Droughtresistance can be determined by any of a number of standard measuresincluding turgor pressure, growth, yield, and the like. In someembodiments, the methods described in the Example section, below can beconveniently used.

EXAMPLES

The following examples are offered to illustrate, but not to limit theclaimed invention.

Example 1 PYR/PYL Modulation of ABA Signaling

Unlike biochemical screens for ABA-binding proteins, genetic analysesfocused on ABA perception have not yet identified proteins resemblingreceptors, suggesting that the receptor(s) may be functionallyredundant, have overlapping functions or cannot mutate to yield viablegametes or seedlings (P. McCourt, Annual Review of Plant Physiology andPlant Molecular Biology 50, 219 (1999)). As a complementary approach, wehave pursued a chemical genetic strategy in plants (Y. Zhao et al., NatChem Biol 3, 716 (2007)). This approach can be advantageous fororganisms with highly redundant genomes, because the variableselectivity of small molecules can cause phenotypes not revealed bysingle gene mutations (N. Raikhel, M. Pirrung, PLANT PHYSIOLOGY 138, 563(2005); S. Cutler, P. McCourt, Plant Physiol. 138, 558 (2005)). Forexample an antagonist with low selectivity can perturb the function ofan entire protein family (as seen with microtubule antagonists), whilean agonist with high selectivity may illuminate the function of anindividual member of normally redundant receptors, as we describe herewith pyrabactin 3 (FIG. 1A).

Pyrabactin is a Seed-Selective ABA Agonist

As part of an earlier effort, we identified a germination inhibitornamed pyrabactin (Y. Zhao et al., Nat Chem Biol 3, 716 (2007)). Byexamining the sensitivity of multiple wild accessions to pyrabactin, wefound that the Cold Spring Harbor Lab wild type, which isABA-hypersensitive and hyperdormant, is also hypersensitive topyrabactin, but not an inactive analog apyrabactin 4 (FIG. 1A). Thissuggested that pyrabactin might act through the ABA response pathway. Totest this hypothesis, we examined the pyrabactin sensitivity of mutantlines with altered ABA signaling, biosynthesis or gibberellic acid (GA)perception. We found that ABA perception, but not biosynthesis, mutantsaffect pyrabactin sensitivity (FIG. 1B). Additionally an rgl2-1 mutantline, which does not require GA during germination (S. Lee et al., GenesDev. 16, 646 (Mar. 1, 2002, 2002)), has normal pyrabactin sensitivity(FIG. 1B). Together, these observations suggest that pyrabactin inhibitsgermination by activating the ABA signaling pathway, rather than bymodulating ABA or GA biosynthesis.

We next performed microarray experiments to evaluate the similarity ofthe transcriptional responses induced by ABA and pyrabactin treatments.For microarray, tissue was prepared and RNA extracted from Columbia wildtype seeds sown on 0.5×MS media (˜2500 seeds per 150 mm plate)containing either 1 μM ABA, 25 μM pyrabactin, 25 μM 2,4-Dinitrophenol(DNP), 1 μM cycloheximide, 2 μM methotrexate or 1% DMSO control plates(all chemicals are dissolved in DMSO). The concentrations utilized forthese experiments were normalized for germination inhibition activity bydose curve analyses, i.e. the amount of both compounds required toensure 100% inhibition of germination when scored 3 dayspost-imbibition. ABA (±stereoisomers), DNP, cycloheximide andmethotrexate were purchased from Sigma Aldrich. Seeds were stratifiedfor 4 days and then incubated in the dark at room temperature for 24hours. Seeds were collected and frozen in liquid nitrogen, then groundto fine powder form with frozen mortar and pestle, after which total RNAwas extracted using the RNAqueous kit (Ambion; Austin, USA) for thefirst set of replicate samples. Subsequent RNA extractions wereperformed using the phenol-chloroform extraction protocol, as describedby (Y. Suzuki, T. Kawazu, H. Koyama, Biotechniques, 37, 542 (October,2004)). For each sample of total RNA, 1 μl of RNA was quantified in 99μl 10 mM Tris-Cl (pH 7.4) by the GeneQuant RNA/DNA Calculator (GEHealthcare Bio-Sciences Corp.; New Jersey, USA), where absorbancemeasurements were taken at 260 nm and 280 nm. Purity of the RNA wasassessed by OD₂₆₀/OD₂₈₀ ratios (only ratios between 1.7 and 2.2 wereused), while quality of the RNA was assessed by gel electrophoresis.Total RNA samples were converted to biotin-labeled cRNA using oligo-dTpriming as described by the manufacturer (Enzo kit; Affymetrix; SantaClara, USA) and hybridized to 22K ATH1 Affymetrix microarrays at theCAGEF (University of Toronto). Duplicate biological replicate sampleswere hybridized for DNP, cycloheximide and methotrexate, triplicate forcontrol and quadruplicate samples were hybridized for, pyrabactin andABA treatments. Probe sets with expression signals called present ormarginal by the statistical algorithms applied to the microarrays asdescribed as described for the GCOS/MAS5.0 algorithm (Affymetrix; SantaClara, USA). Significance Analysis of Microarrays was used to identifyprobe sets that are significantly regulated by treatments using unloggeddata, with a false discovery rate (FDR) at about 5%. Average transcriptlevels were compared to control values to compute fold-change, which wasin turn log₂ transformed and used to compute Pearson CorrelationCoefficients between experiments.

We first examined seeds treated with both compounds for 24 hours. Due toinhibitory effects on seedling development, any two germinationinhibitors will share some common responses; we therefore used apreviously defined set of germination responsive transcripts (G. W.Bassel et al., Plant Physiol 147, 143 (2008)) to minimize developmentaleffects in our comparisons. 1225 probe sets were identified asresponsive to either ABA or pyrabactin using SAM analysis (V. G. Tusher,R. Tibshirani, G. Chu, Proc. Nat'l. Acad. Sci. USA 98, 5116 (2001)),after removal of 403 germination-regulated transcripts. Scatter plotscomparing a probe's responsiveness to pyrabactin and ABA demonstratehighly correlated responses (r=0.98; FIG. 1C), consistent with thehypothesis that pyrabactin activates ABA signaling. As a control, wealso profiled the effects of the three germination inhibitors (G. W.Bassel et al., Plant Physiol 147, 143 (2008)) cycloheximide,methotrexate and 2,4-dinitrophenol, and observed much weakertranscript-response correlations when compared to ABA treatments(r=0.36, 0.73 and 0.81 respectively; cycloheximide shown in FIG. 1D).This demonstrates that an indirect developmental effect is notsufficient to account for the ABA-like transcriptional effects ofpyrabactin.

To establish if pyrabactin is a general ABA agonist, we examined itsactivity in seedlings treated with either compound for 24 hours, whichshowed that pyrabactin induces a greatly muted ABA response (r=0.72) inseedling tissues (FIG. 1E). For seedling microarray experiments,Columbia wild type seeds were surface sterilized and sown on 0.5×MS,0.6% (w/v) agar plates (15 mg seeds, 25 ml media per 150 mm plate),followed by stratification for 4 days at 4° C. and grown under 24-hlight at room temperature for 9 days. 40 seedlings were then transferredto either DMSO control, 10 μM ABA or 33 μM pyrabactin plates andreturned to the growth environment for another 24 hours, after whichtotal RNA was extracted using the method described above. Triplicatesamples were hybridized per treatment. The concentrations used forseedling experiments were based on concentrations of ABA or pyrabactinthat are required to inhibit primary root growth by equivalent amounts,i.e. they were normalized to a measure of bioactivity. In theseexperiments, 57 transcripts responded significantly to both pyrabactinand ABA, suggesting that pyrabactin can induce aspects of an ABAresponse in seedlings. However, since 3021 transcripts in thisexperiment showed a significant response to ABA, but not pyrabactin, weconclude that pyrabactin acts with greater selectivity for the seedpathway in comparison to ABA. Pyrabactin does agonize ABA responses invegetative tissues.

PYR1, a START Protein, is Necessary for Pyrabactin Action

To dissect pyrabactin's mechanism of action, we isolated a collection of16 pyrabactin insensitive mutant lines from a screen of ˜450,000 EMSmutagenized M2 seed. Surface sterilized EMS seeds were sown on 0.33×MSmedia containing 25 μM pyrabactin (50 mg seeds per 150 mm plate). Seedswere stratified for 4 days at 4° C. and grown under constant light for 4days at room temperature, after which plates were scored for mutantsresistant to the germination inhibition effect of pyrabactin. Seedlingswith fully expanded cotyledons were considered resistant, and allmutants identified as resistant were then retested in the nextgeneration to identify true mutants. The strong pyr1-7 allele was usedto map Pyr1 using a mapping population of ˜400 plants (created fromprogeny of a cross to Ler). This delimited Pyr1 to an ˜150 Kb intervalcontaining 12 genes. The identity of Pyr1 was first suggested aftersequencing the 12 genes in this interval and identifying a stop codon inAt4g17870 (Pyr1).

After this, the Pyr1 coding sequence for 14 of the 16 mutations isolatedwere sequenced and 12 independent strains were determined by map basedcloning and sequencing to contain mutations in the same locus,PYRABACTIN RESISTANCE 1 (Pyr1). Pyr1 encodes a protein that is a memberof the START/Bet v 1 superfamily whose members share a conservedligand-binding helix-grip architecture (L. M. Iyer, E. V. Koonin, L.Aravind, Proteins: Structure, Function, and Genetics 43, 134 (2001); C.Radauer, P. Lackner, H. Breiteneder, BMC Evol Biol 8, 286 (2008)). PYR1resides in a Bet v 1 subfamily similar to bacterial polyketidesynthases/cyclases and other non-enzymatic proteins (C. Radauer, P.Lackner, H. Breiteneder, BMC Evol Biol 8, 286 (2008)). There are 13genes in the Arabidopsis genome that show significant similarity to Pyr1in BLAST searches, which we have named PYL1-PYL13 (for PYR1-Like; theirAGIs are listed in Table 1). The pyrabactin insensitive pyr1 alleles weisolated are predicted to produce a variety of defects in PYR1,including truncations and non-conservative amino acid substitutions(FIG. 2A). Transformation of a 35S::GFP-PYR1 expression construct intothe strong pyr1-1 mutant line restores seed pyrabactin sensitivity (FIG.2C), which provides further support that PYR1 is necessary forpyrabactin action. None of the pyr1 alleles isolated show strong ABAinsensitivity, which as we describe below, is explained by the action ofredundant Pyr1 relatives (including, but not limited to Pyl1,2,4). Byquerying public microarray databases (M. Schmid et al., Nat Genet. 37,501 (2005); K. Nakabayashi, M. Okamoto, T. Koshiba, Y. Kamiya, E.Nambara, Plant J 41, 697 (March 2005); H. Goda et al., Plant J 55, 526(August, 2008); D. Winter et al., PLoS ONE 2, e718 (2007); Y. Yang, A.Costa, N. Leonhardt, R. S. Siegel, J. I. Schroeder, Plant Methods 4, 6(2008)) it is clear that Pyr1 mRNA is expressed highly in seeds andguard cells and is responsive to ABA (FIG. 2B), consistent with a rolefor PYR1 in ABA signaling.

TABLE 1 Members of PYR/PYL family and corresponding Arabidopsis GenomeInitative (AGI) annotations. Gene AGI Pyr1 AT4G17870 Pyl1 AT5G46790 Pyl2AT2G26040 Pyl3 AT1G73000 Pyl4 AT2G38310 Pyl5 AT5G05440 Pyl6 AT2G40330Pyl7 AT4G01026 Pyl8 AT5G53160 Pyl9 AT1G01360 Pyl10 AT4G27920 Pyl11AT5G45860 Pyl12 AT5G45870 Pyl13 AT4G18620

PYR/PYL Proteins Bind PP2Cs in Response to ABA

Given that PYR1 is necessary for pyrabactin action and is a predictedligand-binding protein, we hypothesized that pyrabactin agonizes ABAsignaling by inducing a protein-protein interaction between PYR1 and adownstream effector. To test this, ˜2 million prey cDNA clones werescreened against a PYR1 Y2H bait construct on media containing 10 μMpyrabactin. To create the PYR1 Y2H bait construct, the Pyr1 open readingframe was PCR amplified from genomic DNA and cloned to pGem-T easyvector (Promega). After sequence confirmation, the Pyr1 ORF was thencloned in-frame between EcoRI and SalI sites of the pBD-GAL4 Cam vector(Stratagene) and transformed into yeast strain Y190. For the screen, anetiolated seedling cDNA library (J. Kim, K., Harter, A., Theologis, ProcNatl Acad Sci US A 94, 11786 (Oct. 28, 1997)) (ABRC stock CD4-22) wasused. The cDNA library was first converted from phage to plasmid DNA,yielding 7.6×10⁷ transformants. Plasmid DNA prepared from library wasthen used to transform Y190 as described in the GAL4 Two-Hybrid systemmanual (Stratagene). For each screen, 40 μg of prey plasmid wastransformed into 1 ml of competent Y190 cell harboring bait constructand then grown on SD agar plates lacking His, Leu, and Trp, butcontaining 15 mM 3-AT and 10 μM pyrabactin. After 4 days incubation at30° C., well-grown colonies were rescued and interactions validatedusing filter lift assay or chloroform-agarose overlay method and X-Galstaining. This identified two pyrabactin-dependent hits which sequencingdetermined encoded cDNAs for the PP2C HAB1, a close relative of thewell-characterized ABA response factor ABI1(A. Saez et al., The PlantJournal 37, 354 (2004); N. Leonhardt et al., THE PLANT CELL 16, 596(2004)). Next, Y2H strains expressing an AD-HAB1 fusion protein and aBD-PYR1 fusion protein were grown on plates and tested for interactionsin response to various compounds, all at 10 μM except forepi-brassinolide (50 nM) and dimethyl sulfoxide (DMSO) (carrier solvent,1%). When the pyrabactin-responsive PYR1-HAB1 Y2H strains were tested on(+)-ABA, strong interactions were observed by X-gal stain, but neither(−)-ABA, kinetin, 2,4-D, Gibberellic acid (GA), epi-brassinolide (BR),methyl jasmonate (meJA) or apyrabactin showed activity (FIG. 3A). Thus,PYR1 interacts with HAB1 in a (+)-ABA dependent fashion.

To see if ABA and pyrabactin responsiveness is unique to PYR1, we tested11 of the 13 PYL proteins as described above, using Y2H strainsexpressing an AD-HAB1 fusion protein and a BD-PYR/PYL fusion protein(listed at the left of FIG. 3A). BD-PYR/PYL fusion proteins wereconstructed in the same manner as for BD-PYR1 above. This assay showedthat PYL1-PYL4 interact with HAB1 in an ABA-stimulated manner (FIG. 3A).Ligand-selective interactions are also observed for pyrabactin, whichpromotes interactions between HAB1 and PYR1, PYL1, or PYL3 (FIG. 3A). Ofthese, only Pyr1 is highly transcribed in seeds, which likely explainswhy mutations in Pyr1 cause the seeds to be insensitive to pyrabactin.PYL2-PYL4 respond to both (+)-ABA and (−)-ABA (FIG. 3A), suggesting thatthey could be involved in both (+) and (−)-ABA responses. Notably, theremaining PYLs tested in the yeast two hybrid assay show constitutiveinteractions with HAB1, suggesting they may have different thresholdsfor interaction with the PP2Cs from PYR1 and PYLs 1 to 4. However theinteractions of PYLs 5-12 with the PP2Cs are indicative that the entireprotein family is likely to share a similar mechanism of actioninvolving PP2C modulation, as we describe below. Thus, we conclude thatentire family modulates ABA responses via PP2C interactions.

To investigate the ABA/pyrabactin responses further, we used the Y2Hassay as described above to examine three substitution mutant proteinsthat cause strong pyrabactin insensitive phenotypes in plants. Two ofthe mutants tested, PYR1^(S152L) and PYR1^(P88S), greatly reduce ABAinduced PYR1-HAB1 interactions, while the PYR1^(R157H) mutation does notaffect the interaction (FIG. 3B). HAB1 possesses genetic redundancy withABI1, ABI2 and other related PP2Cs (T. Yoshida et al., PLANT PHYSIOLOGY140, 115 (2006)). We therefore tested ABI1 and ABI2 in the Y2H assay,using publicly available sequence validated cDNAs for ABI1 and ABI2(C104649, and U24491 respectively). We observed that PYR1 interacts withwild type ABI1 and ABI2, but not the ABA insensitive proteinABI2^(G168D) encoded by abi2-1 (FIG. 3C). Thus, residues important toPYR1 and PP2C function in planta are important for the ABA responsereconstituted in yeast. These in vivo interactions between PYR1 and PP2Clikely occur in the cytoplasm and nucleoplasm, as suggested by thelocalization pattern observed for GFP-PYR1 (FIG. 4).

PYR/PYL Proteins Act Redundantly in ABA Signaling

To examine whether the ABA-responsive PYL proteins act redundantly withPYR1 in ABA signaling, we isolated homozygous insertion alleles forPYL1, 2 and 4 from public insertion-allele collections (seedstrains=Salk_(—)054640, GT_(—)2864, Sail_(—)517_C08 respectively) (J. M.Alonso et al., Science 301, 653 (2003); A. Sessions et al., THE PLANTCELL 14, 2985 (2002); V. Sundaresan et al., Genes and Development 9,1797 (1995)). The homozygous insertion lines and pyr1-1 were crossed tocreate pyr1-1:pyl2-1 and pyl1-1:pyl4-1 heterozygous lines, which werethen crossed to one another. ˜70 progeny from this cross were genotypedby PCR to identify lines heterozygous for all 4 mutations, and 2 plantswere identified. To assess if these lines segregated ABA insensitiveplants, the F2 seed from a quadruple heterozygous plant were germinatedon 0.7 μM (+)-ABA. Extensive variation in germination and growth wasobserved, and the most ABA-resistant seedlings were selected from ˜1000seed and genotyped by PCR and sequencing. None of the homozygous singlemutant parents showed marked ABA insensitivity, but both a triple(pyr1-1, pyl1-1, pyl4-1) and quadruple (pyr1-1, pyl1-1, pyl2-1, pyl4-1)mutant line showed ABA insensitivity. The root and germination responsesof the quadruple and triple mutants lines were examined in comparison toabi1-1, the strongest ABA-insensitive mutant isolated to date. Forgermination assays, seeds were stratified on plates containing (+)-ABAon 0.33×MS for 4 days at 4° C. and then germinated at 23° C. in the darkfor 3 days at 90% RH. Seeds showing radicals ½ seed length or longerwere scored as positive for germination. To investigate root growth,seeds were allowed to first germinate on MS plates after 4 days ofstratification and then transferred to germinate at 23° C. in darknessat 90% RH. 48 hours post imbibition, seedlings showing radical emergencewere transferred to (+)-ABA containing or control plates, grownvertically for 4 additional days in the dark and then new root growthmeasured. In germination assays, the quadruple mutant was moreinsensitive than the triple, but both exhibited a weaker phenotype thanabi1-1 (FIG. 5A). In root growth assays, the quadruple and triple mutantlines both showed greater ABA insensitivity than abi1-1 (FIG. 5B). Thequadruple mutant line also exhibits defects in ABA-induced geneexpression. Quantitative RT-PCR experiments were conducted as describedpreviously (H. Fujii, et al., Plant Cell, 19, 485 (2007)) using taqmanprobes identical to those described by Fujii et al. Briefly, 7 day oldseedlings grown under continuous illumination on 0.3×MS plates weretransferred to 0.3×MS media containing carrier solvent (0.1% DMSO) or100 μM (+)-ABA for 5 hours, after which total RNA was isolated usingQiagen plant RNeasy isolation kit. 5 μg total RNA was used per 20 μLfirst strand cDNA synthesis reaction using SuperScript ReverseTranscriptase. The reactions were diluted to 100 μl with TE and 1.5 μlof this was used in 15 μL qRT-PCR reactions using taqman probesdescribed previously (6). Values shown are the average of triplicatemeasurements. Quadruple mutants exhibit decreased transcription of theABA-responsive genes RD29 (FIG. 2D), NCED3 (FIG. 2E), and P5CS1 (FIG.2E) in the presence of (+)-ABA. These experiments show that PYL1, PYL2and PYL4 function redundantly with PYR1 in the control of ABA-inducedgene expression and germination and root responses to ABA.

In Vitro Reconstitution of ABA Perception: ABA and PYR1 Inhibit PP2CActivity

To explore the functional implications of the PYR1-PP2C interaction, weexamined if an ABA response could be reconstituted in vitro. RecombinantGST-HAB1, GST-ABI1 and GST-ABI2 were expressed in E. coli and tested forligand-dependent interactions with 6×His-PYR1 in pull-down assays.Purified 6×His-PYR1 and GST-HAB1 (20 and 100 μg respectively, 8 μM PYR1final concentration), were combined in 100 μl TBS containing 10 μM(+)-ABA or 1% DMSO for negative control. The reaction was incubated for90 minutes at RT and 5 μl of PrepEase (USB) His-tagged proteinpurification resin was added. The resin and reaction mixture wasincubated 30 min at RT with gentle shaking at 5 min intervals. The resinwas washed five times with TBS containing 10 μM (+)-ABA. After the finalwash, the bound protein was eluted in 20 μl SDS-PAGE buffer, boiled for5 minutes and centrifuged. 5 μl of eluate was analyzed on SDS-PAGE. Forpull-downs with ABI1 and ABI2, crude lysates were used in a similarmethod, except purified PP2C was replaced with cleared E. coli lysates.The amount of lysate added was determined by SDS-page analysis to yield˜100 μg PP2C, such that the same stoichiometry was used as in assaysusing purified proteins. We found that both (+)-ABA and pyrabactinpromote PP2C interactions with PYR1; however PYR1^(P88S) is insensitivein this assay (FIG. 6A).

Since ABI1 and relatives are negative regulators of the ABA signalingpathway, we hypothesized that the function of the ABA-promoted PYR1-PP2Cinteraction was to inhibit phosphatase activity and remove a negativeinput into the pathway, which would then promote signaling. To test thishypothesis, we examined the effects of (+)-ABA on PP2C enzyme kineticsusing recombinant GST-HAB1, 6×His-PYR1 or 6×His-PYR1^(P88S) using thephosphatase substrate pNPP. The ORF of Arabidopsis HAB1 was amplified byPCR from a pUni clone obtained from the ABRC and cloned into pGex-2T tocreate a GST-HAB1 fusion protein. Both constructs were transformed intoBL21[DE3]pLysS. For expression, cells harboring pGex-GST-HAB1 were grownovernight in 20 ml LB and then inoculated to 700 ml media containing 1mM MnCl₂ and continued incubation with shaking at RT for 8 hr. Proteinexpression was then induced by addition of IPTG to final concentrationof 0.5 mM, and cells were cultured overnight at RT. Cells were thenharvested by centrifugation at 4500 rpm for 20 min, resuspended in 10 mlTBS containing 10 mM MnCl₂. Cells were stored at −80° C. To preparecleared lysates, cells were freeze-thawed twice and the lysate'sviscosity reduced by shearing. The lysate was then spun at 12000×g for10 min to yield the final cleared lysates. This was applied to 1 ml ofimmobilized glutathione column, washed with 20 ml of TBS and boundprotein then eluted with 20 mM reduced glutathione. The eluate wasdialyzed against TBS containing 10 mM MnCl₂. MnCl₂ was used throughpurification steps and found to be critical for recovery of highlyactive HAB1 protein, as described previously for other PP2Cs (C. C.Fjeld, J. M. Denu, J Biol Chem, 274, 20336 (Jul. 16, 1999)). The PYR1and PYR1^(P88S) coding sequences were amplified by PCR from genomic DNAof wild type or the pyr1-3 mutant respectively and cloned into pET28 toproduce various 6×His-PYR1 proteins, which were validated by sequencing.For 6×His-PYR1 and 6×His-PYR1^(P88S) protein expressions, 20 ml of anovernight culture was inoculated to 700 ml LB and was grown foradditional 3 hours at 37° C. with shaking. Protein expression wasinduced by addition of IPTG to 1 mM. Cells were harvested 5 hr later bycentrifugation for 15 min at 5000×g and the pellet was resuspended in 5ml of the Buffer A (50 mM NaH₂PO₄, 300 mM NaCl) containing 10 mMimidazole, pH 8.0). Cells were stored at −80° C. before purification.After thawing, cells were sonicated on ice five times for 30 sec with 30sec resting intervals. A cleared lysate was obtained aftercentrifugation at 12,000×g for 10 min and applied to 1 ml of Ni-NTAcolumn (Qiagen) and washed with 20 column volumes of Buffer A containing30 mM imidazole. Bound protein was eluted with 10 ml of Buffer A with100 mM imidazole. The elutate was dialyzed against TBS. For the pNPPassay, initial reaction velocities for GST-HAB1 were conducted using thesynthetic phosphatase substrate pNPP. Reactions contained 1 μM GST-HAB1,1.5 μM 6×His-PYR1 or 6×His-PYR1^(P88S) and a reaction buffer consistingof 33 mM Tris-OAc, pH 7.9, 66 mM KOAc, 0.1% BSA, 25 mM Mg(OAc)₂, 50 mMpNPP and varying (+)-ABA concentrations. Reactions were initiated by theaddition of assay buffer to protein/ABA mixes. Immediately after mixing,reactions were monitored for hydrolysis of pNPP at A405 t˜10 secondintervals over 20 minutes using a Wallac plate reader. Reactionprogressions were plotted, initial velocities calculated and convertedto specific activities using a standard curve for 4-nitrophenol made inthe same buffer system volumes/plate reader used for enzymatic reactionmeasurements. These experiments show that (+)-ABA acts as a potentinhibitor of HAB1 phosphatase activity (IC₅₀=0.18 μM) in the presence ofPYR1, but not PYR1^(P88S) (FIG. 6B).

Similarly, ABA displays saturable inhibition of HAB1 PP2C activity inthe presence of recombinant PYL4. A PYL4 6×His-tagged (SEQ ID NO:141)protein was constructed using a public pUni clone. This was recombinedinto the His-tagged expression vector pHB3. The construct was expressedin BL21[DE3] pLysS as described above for PYR1, but the protein formedinclusion bodies, which were solubilized in Buffer B+8 M urea, prior topurification. The protein was purified under denaturing conditions usingNi-NTA resin according to manufacturer's instructions. After binding ofprotein to resin, the column was washed with 20 volume of Buffer B(pH6.3) and protein eluted using Buffer A (pH4.5). The eluted proteinwas dialyzed slowly from TBS containing 2 M urea, 10 mM DTT into TBScontaining 1 mM DTT over three days, gradually lowering the ureaconcentration over time. The activity of refolded PYL4 was validatedusing in vitro pull down assays developed for PYR1, where it was shownthat PYL4 binds HAB1 in response to ABA. For the PP2C assays,recombinant PYL4 (refolded from inclusion bodies) and HAB1 were used.When phosphatase activity was measured for GST-HAB1 using thephosphatase substrate pNPP, we found that (+)-ABA inhibits HAB1phosphatase activity in the presence of PYL4 (FIG. 6C). Thus, PP2Cinhibition is a primary ABA-response that can be reconstituted in vitrowith only proteins.

DISCUSSION

We have shown that PYR1 has the properties expected of an ABA receptorand that it binds to and inhibits PP2C activity when ligand is present.In contrast to previously identified ABA binding proteins (P. McCourt,R. Creelman, Current Opinion in Plant Biology 11, 474 (2008)), PYR1interacts directly with core components of the ABA signaling pathway.ABI1 interacts with at least one positively acting factor in the ABAresponse pathway (R. Yoshida et al., Journal of Biological Chemistry281, 5310 (2006)). It may therefore be that the role of ABI1/AHG1 classPP2Cs in the absence of a signal is to repress the action of positivelyacting factors. In this model, ABA functions at the apex of a negativeregulatory pathway and the PP2Cs control signal output through theirdirect targets. This imbues the PP2Cs with a critical role incontrolling the selectivity of signal-output, which could explain theextensive diversification of the PP2C gene family in plants relative toanimals (A. Schweighofer, H. Hirt, I. Meskiene, Trends in Plant Science9, 236 (2004)). Based on the interaction of PP2Cs with SnRK2 proteinsand the critical role of SnRK2s for ABA signaling (FIG. 7) we haveproposed the following model for ABA action in which ABA and PYR/PYLsinhibit the PP2Cs, which in turn relieves repression of positivefactors, such as the SnRK2s. This in turn allows the positive SnRK2kinases to modulate activity of downstream factors via phosphorylation.

Our experiments show that at least 12 of the 14-members in the PYR/PYLgene family bind to PP2Cs, and some members such as PYL2s, 3 and 4enable yeast cells to respond to the unnatural stereoisomer (−)-ABA. Webelieve the entire family are ABA receptors and that some may also be(−)-ABA receptors. This hypothesis is consistent with earlierconclusions that both stereoisomers act through the same signalingpathway (E. Nambara et al., Genetics 161, 1247 (July, 2002)).

PYR1 is unable to bind to the proteins encoded by abi1-1 and abi2-1,which both contain mutations in glycines near one of the two conservedPP2C metal binding sites. These mutations lower, but do not abolish,PP2C activity (F. Gosti et al., The Plant Cell 11, 1897 (1999); N.Robert, S. Merlot, V. N′Guyen, A. Boisson-Dernier, J. I. Schroeder, FEBSLetters 580, 4691 (2006)) and a second site mutation that completelyabolishes abi1-1's catalytic activity suppresses its dominant phenotype(F. Gosti et al., The Plant Cell 11, 1897 (1999)). Together with ourobservations on defective PYR1 interactions, these data suggest a modelwhere the dominance of the abi1-1 and abi2-1 mutations stems from theirability to escape negative regulation by the PYR/PYL proteins. In thismodel, a major function of ABA is to lower ABI1/AHG1 class PP2C activityvia PYR/PYL proteins, but this does not occur properly in the abi1-1 andabi2-1 mutant lines, which retain sufficient PP2C activity after ABAperception to disrupt signal transduction.

The regulation of PP2Cs is poorly understood with respect to otherphosphatase classes, which is surprising given their important roles inmammals, worms, flies and yeast (G. Lu, Y. Wang, Clinical andExperimental Pharmacology and Physiology 35, 107 (2008)). Ourobservations provide a new mechanism for receptor-mediated regulation ofPP2C activity. Although the precise mechanism of PP2C inhibition by PYR1is unknown, the PYR1^(R157H) mutation is able to separate ligandperception from downstream functions in vivo. This residue may thereforeplay a critical role in steps that lead to inhibition of PP2C activityafter signal perception. Regardless of the precise details of PP2Cinhibition, the novel regulatory mechanism discovered suggests that itmay be worth investigating receptor-mediated PP2C regulation in othermodels, given the dearth of regulatory factors for these vitalphosphatases.

The ABA signaling pathway has been the subject of genetic analysis foralmost 30 years, but the PYR/PYL proteins never emerged as factorsnecessary for an ABA response in genetic screens. In hindsight, this isnow obvious due to the necessity of a triple mutant to observe anABA-insensitive phenotype. When using pyrabactin as a synthetic agonistof the pathway however, Pyr1 was identified with ease. The reason forthis is due to pyrabactin's selectivity for a subset of the entirereceptor family, which enabled us to bypass the genetic redundancy thatobscures an ABA phenotype in single mutant lines. Thus, our resultsdemonstrate the power of the chemical genetic approach to revealphenotypes for normally redundant genes. Because plant genomes arehighly redundant, we expect that small molecule approaches will providea powerful addendum to classical genetic analysis.

Example 2 Screens for Agonists of PYR/PYL

We next investigated whether other compounds besides ABA and pyrabactincould act as agonists of PYR/PYL proteins. Yeast two hybrid strainsexpressing ABA-receptors and type 2 C protein phosphatases in theappropriate vectors can be used to monitor activation of ABA receptors.These yeast strains therefore create a facile screening system for theidentification of cell permeant compounds that act as ABA agonists, i.e.compounds that promote binding of PYR/PYL family members to theirprotein phosphatase targets. When PYR/PYL proteins are bound to PP2Ctargets in the yeast two hybrid context, a reporter gene is activatedwhich, depending on strains used, can lead to expression of a reporterconstruct such as the LacZ/B-galactoisidase marker or to a nutritionalreporter gene that enables growth on auxotrophic media.

To conduct these agonist assays, screening compounds are added tomicrotiter wells and appropriate yeast growth media are added. The wellsare then seeded with PYR/PYL-PP2C strains and agonist activity ismonitored after growth of the strains on the chemical-containing medium.Numerous approaches can be used to monitor activation including simplegrowth (via restoration of expression of a nutritional reporter gene) ofcolorimetric X-gal assays, which are well known in the art. Analternative screening method, called the “Halo Assay,” can also be usedto identify agonists. In this assay, yeast strains can be embedded insuitable growth medium containing agarose and chemicals can be spottedonto plates using a pin replicator. The growth medium, lacking anutrient needed for growth, prevents yeast growth unless one of thescreening chemicals supplied enters the yeast cell and activates thePYR/PYL receptors, which results in expression of the nutritional markergenes in the yeast two hybrid strain. Activated cells appear as regionsof cell growth and can be easily identified by visual inspection.

Using a combination of the conventional and halo assays as describedabove, 65,000 screening compounds were tested for activation of PYR1,PYL2, PYL3 or PYL4 expressing yeast two hybrid strains. Hit compoundsthat activated any of the yeast strains were retested on all 4 yeaststrains and activity assessed qualitatively using X-gal staining assays.This led to the identification of the compounds shown in FIG. 8.Estimates of the relative activity of each of these compounds on thePYR/PYL receptors PYR1, PYL1, PYL2, PYL3, and PYL4 is depicted in FIG.8. We note that the PYL3 yeast strain used in these screening assays isexceptionally sensitive to ABA, and therefore the estimate of therelative activity of ABA or other compounds on the PYL3 receptor may berefined by later performing in vitro phosphatase assays, describedbelow.

As a further validation of hit compounds identified in the yeasttwo-hybrid assay, we utilized in vitro PP2C assays conducted in thepresence of recombinant PYR/PYL receptor proteins PYR1, PYL1, PYL2, orPYL3 and the PP2C HAB1. Recombinant proteins were made as describedabove in Example 1. Phosphatase assays using the phosphatase substratepNPP were performed as described in Example 1. As demonstrated by theIC50 values, we found that compound 7653159, which is the same compoundas compound 7 in FIG. 8, is a potent agonist of PYR1 and PYL1 inhibitionof HAB1 but is not an agonist for PYL2 or PYL3 (FIG. 9). Similarly,compound 6655097, which is the same compound as compound 6 in FIG. 8, isa potent agonist of PYR1 and PYL1 inhibition of HAB1 but is not anagonist for PYL2 or PYL3 (FIG. 9). Compound 7561035, which is the samecompound as compound 9 in FIG. 8, is a potent agonist of PYL2 and PYL3inhibition of HAB1 but is not an agonist for PYR1 or PYL1 (FIG. 9).

Example 3 Phenotypic Analysis of PYR/PYL Overexpression andLoss-of-Function Mutant Plants

Abscisic acid is a multifunctional phytohormone involved in a variety ofphyto-protective functions including bud dormancy, seed dormancy and/ormaturation, abscission of leaves and fruits, and response to a widevariety of biological stresses (e.g. cold, heat, salinity, and drought).ABA is also responsible for regulating stomatal closure by a mechanismindependent of CO₂ concentration. Because PYR/PYL receptor proteinsmediate ABA signaling, these phenotypes can be modulated by modulatingexpression of PYR/PYL. However, as discussed above, experiments withsingle, triple, and quadruple Pyr/Pyl mutant plants demonstrate that PYLreceptors PYL1, 2 and 4 function redundantly with PYR1 in the control ofgermination and root responses to ABA function. In these experiments, weasked whether other PYR/PYL receptors function redundantly with PYR1 inthe control of plant phyto-protective functions such as flowering time,stature, chlorophyll content, and wiltiness. We used the pyr1; pyl1;pyl2; pyl4 quadruple mutants as described above in Example 1 to test theeffect of loss of function of multiple PYR/PYL receptors on thesephyto-protective functions. We found that pyr1; pyl1; pyl2; pyl4quadruple mutants exhibit defects in flowering time, stature, andwiltiness (FIG. 10). Relative to a control Arabidopsis plant, pyr1;pyl1; pyl2; pyl4 quadruple mutants flower early, are smaller in stature,and are very wilty. We also examined the effect on phyto-protectivefunctions from overexpressing the PYR/PYL receptor PYL4. We generatedtransgenic Arabidopsis plants expressing GFP-PYL4 under the control ofthe high expression promoter Rbcs, and found that plants thatoverexpress PYL4 exhibit defects in flowering time, stature, wiltiness,and the chlorophyll content of the plants; relative to control plants,these PYL4-overexpressing plants flower later, are darker green, andless wilty (FIG. 10). These results demonstrate that PYR/PYL receptorsmodulate a wide variety of ABA-mediated activities in plants.

Example 4 Screens of Plant Extracts for PYR/PYL Agonists

The yeast strains expressing PYR/PYL receptors and type 2 C proteinphosphatases were also used to screen HPLC-fractionated plant extractsfor the presence of endogenous compounds that activate PYL/PYL receptorsPYR1, PYL2, PYL3, and/or PYL4. HPLC fractionation of extracts was usedto identify compounds different from abscisic acid (the known agonist).This led to the identification of a PYL3/PYL4 selective agonist inextracts made from Hypericum perforatum aerial tissues. Purification ofthe agonist was achieved via multiple rounds of chromatographicseparation coupled to yeast two hybrid assays that informed thefractions to move forward at each step of the purification. Thestructure of the purified agonist was deduced by X-ray crystallographyof crystalline purified agonist. This revealed the compound to be thepreviously known compound artemisinic acid. This compound has not beenreported outside of the genus Artemisia (Asteraceae) and our isolationof this compound from Hypericum (Clusiaceae) suggests the compound mayhave widespread occurrence in plants, consistent with a functionallyimportant role to plant physiology. Several related compounds wereobtained from commercial sources and also found to possess PYL3/PYL4selective agonist activity (FIG. 12). Following a similar approach tothat described above for artemisinic acid, a second naturally occurringABA agonist was identified from seeds of Cola accumulata and identifiedby 2D-NMR as a previously undescribed derivative of alpha-copaene,copaenoic acid (FIG. 12).

It is understood that the examples and embodiments described herein arefor illustrative purposes only and that various modifications or changesin light thereof will be suggested to persons skilled in the art and areto be included within the spirit and purview of this application andscope of the appended claims. All publications, patents, and patentapplications cited herein are hereby incorporated by reference in theirentirety for all purposes.

INFORMAL SEQUENCE LISTING SEQ ID NO: 1 xWxxxxxFxxPxxxxxFxxxCSEQ ID NO: 2 Brassica_oleracea {89257688} gb|ABD65175.1|mpsqltpeerselaqsiaefhtyhlgpgscsslhaqrihappeivwsvvrrfdkpqtykhfikscsvedgfemrvgctravnvisglpantsterldildderrvtgfsiiggehrltnyksvttvhrfekerriwtvvlesyvvdmpegnseddtrmfadtvvklnlqklatvteamarnagdgsgaqvt SEQ ID NO: 3 Brassica_oleracea {89274227} gb|ABD65631.1|mpseltqeerskltqsisefhtyhlgpgscsslhaqrihappeivwsvvrqfdkpqtykhfikscsveegfemrvgctrdvivisglpantsterldmldderrvtgfsiiggehrlknyksyttvhrfererriwtvvlesyvvdmpegnseddtrmfadtvvklnlqklatvteamarnagdgrgsrettcresfhlitafekqrqiteptvyqnppyhtgmtpeprtstvfieledhrtlpgnitptteehlqrmyqrfwgirqlqrprqsfgerqsi SEQ ID NO: 4 Vitis_vinifera {157341954} 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emb|CAO43790.1|mdphhhhglteeefralepiiqnyhtfepspntctslitqkidapaqvvwpfvrsfenpqkykhfikdctmrgdggvgsirevtvvsglpaststerleilddekhilsfrvvggehrinnyrsvtsvndfskegkdytivlesyivdipegntgedtkmfvdtvvklnlqklavvaitslheneeiadnegpsreislqsetesaergderrdgdgpskacnrnewhcttke SEQ ID NO: 11Oryza_sativa_Japonica_Group {49388537} dbj|BAD25659.1|mephmeralreavaseaerrelegvvrahhtfpaaeraagpgrrptctslvaqrvdaplaavwpivrgfanpqrykhfikscelaagdgatvgsvrevavvsglpaststerleildddrhvlsfrvvggdhrtrnyrsvtsvtefsspsspprpycvvvesyvvdvpegnteedtrmftdtvvklnlqklaavatsssppaagnhh SEQ ID NO: 12Oryza_sativa_Indica_Group {125538682} gb|EAY85077.1|mephmeralreavaseaerrelegvvrahhtfpaaeraagpgrrptctslvaqrvdaplaavwpivrgfanpqrykhfikscelaagdgatvgsvrevavvsglpaststerleildddrhvlsfrvvggdhrtrnyrsvtsvtefsspssppspprpycvvvesyvvdvpegnteedtrmftdtvvklnlqklaavatsssppaagnhh SEQ ID NO: 13 Zea_mays {194695858}gb|ACF82013.1|mpytaprpspqqhsrvlsgggakaashgascaavpaevarhhehaaragqccsavvqaiaapvgavwsvvrrfdrpqaykhfirscrlvgggdvavgsvrevrvvsglpatssrerleildderrvlsfrvvggehrlanyrsvttvheagagagtgtvvvesyvvdvphgntadetrvfvdtivrcnlqslartaerla SEQ ID NO: 14 Vitis_vinifera {157355387}emb|CAO48777.1|mpsnppksslvvhrinspnsittattasaaannhntstmpphkqvpdavsrhhthvvgpnqccsavvqqiaapvstvwsvvrrfdnpqaykhfvkschvvvgdgdygtlrevhvisglpaansterleildderhvlsfsviggdhrlsnyrsvttlhpspsstgtvvlesyvvdippgntkedtcvfvdtivrcnlqslaqiaenaagckrsss SEQ ID NO: 15Nicotiana_tabacum {62867576} emb|CAI84653.1|mppsspdssyllqrissnttpdfackqsqqlqrrtmpipcttqvpdsvvrfhthpvgpnqccsaviqrisapystvwsvvrrfdnpqaykhfvkschvivgdgdygtlrevrvisglpaassterleildderhvisfsvvggdhrlanyrsyttlhpepsgdgttivvesyvvdvppgntrdetcvfvdtivkcnltslsqiavnvnrrkds SEQ ID NO: 16Oryza_sativa_Indica_Group {125528236} gb|EAY76350.1|mpyaavrpspppqlsrpigsgagggkacpavpcevaryhehavgagqccstvvqaiaapadavwsvvrrfdrpqaykkfikscrlvdgdggevgsvrevrvvsglpatssrerlevldddrrvlsfrivggehrlanyrsyttyheaaapamavvvesyvvdvppgntweetrvfvdtivrcnlqslartverlapeaprangsidha SEQ ID NO: 17Oryza_sativa_Japonica_Group {15624049} dbj|BAB68102.1|mpyaavrpspppqlsrpigsgagggkacpavpcevaryhehavgagqcfstvvqaiaapadavwsvvrrfdrpqaykkfikscrlvdgdggevgsvrevrvvsglpatssrerlevldddrrvlsfrivggehrlanyrsyttyheaaapamavvvesyvvdvppgntweetrvfvdtivrcnlqslartverlapeaprangsidha SEQ ID NO: 18Picea_sitchensis {116783434} gb|ABK22940.1|mdiiagfdqlsfrlsgaskqftktgavqylkgeegygewlkeymgryhyhshdgarecrcssvvvqqveapvsvvwslvrrfdqpqvykhfvsncfmrgdlkvgclrevrvvsglpaatsterldildeerhilsfsivggdhrinnyrsittlhetlingkpgtlviesyvldvphgntkeetclfvdtivkcnlqslahvsnhlnsthrcl SEQ ID NO: 19Oryza_sativa_Japonica_Group {115468550} ref|NP_001057874.1|meahveralreglteeeraalepavmahhtfppstttattaaatctslvtqrvaapvravwpivrsfgnpqrykhfvrtcalaagdgasvgsvrevtvvsglpaststerlemldddrhiisfrvvggqhrlrnyrsvtsvtefqppaagpgpappycvvvesyvvdvpdgntaedtrmftdtvvklnlqmlaavaedsssasrrrd SEQ ID NO: 20Oryza_sativa_Japonica_Group {115464439} ref|NP_001055819.1|mpytaprpsppqhsriggcggggvlkaagaaghaascvavpaevarhhehaagvgqccsavvqaiaapydavwsvvrrfdrpqaykhfirscrlldgdgdggavavgsvrevrvvsglpatssrerleildderrvlsfrvvggehrlsnyrsyttyhetaagaaaavvvesyvvdvphgntadetrmfvdtivrcnlqslartaeqlalaapraa SEQ ID NO: 21Vitis_vinifera {157351249} emb|CAO41436.1|mpsslqlhrinnidpttvavaataavnchkqsrtplrcatpvpdavasyhahavgphqccsmvvqttaaalptvwsvvrrfdnpqaykhflkschvifgdgdigtlrevhvvsglpaessterleildderhvlsfsvvggdhrlcnyrsvttlhpsptgtgtvvvesyvvdippgntkedtcvfvdtivkcnlqslaqmsekltnnnrnss SEQ ID NO: 22 Zea_mays {195617008}gb|ACG30334.1|mpclqasspgsmpyqhhgrgvgcaaeagaavgasagtgtrcgandgevpaeaarhhehaapgpgrccsavvqrvaapaeavwsvvrrfdqpqaykrfvrscallagdggvgtlrevrvvsglpaassrerlevlddeshvlsfrvvggehrlqnylsvttvhpspaapdaatvvvesyvvdvppgntpedtrvfvdtivkcnlqslattaeklalaav SEQ ID NO: 23Physcomitrella_patens_subsp._patens {168051209} ref|XP_001778048.1|mqtkgrqadfqtllegqqdlicrfhrhelqphqcgsillqlikapvetvwsvarsfdkpqvykriiqtceiiegdggvgsirevrlvssipatssierleilddeehiisfrvlggghrlqnywsvtslhsheidgqmgtlvlesyvvdipegntreethmfvdtvvrcnlkalaqvseSEQ ID NO: 24 Oryza_sativa_Indica__Group {125543492} gb|EAY89631.1|mpcipasspgiphqhqhqhhralagvgmavgcaaeaavaaagvagtrcgandgevpmevarhhehaepgsgrccsavvqhvaapapavwsvvrrfdqpqaykrfvrscallagdggvgtlrevrvvsglpaassrerleilddeshvlsfrvvggehrlknylsvttvhpspsaptaatvvvesyvvdvppgntpedtrvfvdtivkcnlqslaktaeklaagaraags SEQ ID NO: 25Oryza_sativa_Japonica_Group {115452475} ref|NP_001049838.1|mpcipasspgiphqhqhqhhralagvgmavgcaaeaavaaagvagtrcgahdgevpmevarhhehaepgsgrccsavvqhvaapaaavwsvvrrfdqpqaykrfvrscallagdggvgtlrevrvvsglpaassrerleilddeshvlsfrvvggehrlknylsvttvhpspsaptaatvvvesyvvdvppgntpedtrvfvdtivkcnlqslaktaeklaagaraags SEQ ID NO: 26Medicago_trancatula {217075076} gb|ACJ85898.1|mpspvqfqrfdsntaitngvncpkqiqacryalsslkptvsvpetvvdhhmhvvgqnqcysvviqtinasystvwsvvrrfdypqgykhfvkscnvvasgdgirvgalrevrlvsglpavssterldildeerhvisfsvvggvhrcrnyrsvttlhgdgnggtvviesyvvdvpqgntkeetcsfadtivrcnlqslvqiaekl SEQ ID NO: 27 Zea_mays {195608982}gb|ACG26321.1|mpfaasrtsqqqhsrvatngravavcaghagvpdevarhhehavaagqccaamvqsiaapvdavwslvrrfdqpqrykriirschlvdgdgaevgsvrelllvsglpaessrerleirdderrvisfrvlggdhrlanyrsvttvheaapsqdgrpltmvvesyvvdvppgntveetrifvdtivrcnlqslegtvirqleiaamphddnqn SEQ ID NO: 28Zea_mays {194705858} gb|ACP87013.1|mrernssidgehqrgsssrstmpfaasrtsqqqhsrvatngravavcaghagypdevarhhehavaagqccaamvqsiaapvdavwslvrrfdqpqrykriirschlvdgdgaevgsvrelllvsglpaessrerleirdderrvisfrvlggdhrlanyrsyttyheaapsqdgrpltmvvesyvvdvppgntveetrifvdtivrcnlqslegtvirqleiaamphddnqn SEQ ID NO: 29Physcomitrella_patens_subsp._patens {168019160} ref|XP_001762113.1|mmqekqgrpdfqfllegqqdlicrfhkhellphqcgsillqqikapvqtvwlivrrfdepqvykriiqrcdivegdgyvgsirevqlvssipatssierleilddeehiisfrvlggghrlqnywsvtslhrheiqgqmgtlylesyvvdipdgntreethtfvdtvvrcnlkalaqvseqkhllnsnekpaap SEQ ID NO: 30 Vitis_vinifera {157354734} emb|CAO48052.1|mkvyspsqilaergpraqamgnlyhthhllpnqcsslvvqttdaplpqvwsmvrrfdrpqsykrfvrgctlrrgkggygsvrevnivsglpaeislerldkldddlhymrftviggdhrlanyhstltlhedeedgvrktvvmesyvvdvpggnsagetcyfantiigfnlkalaavtetmalkanipsgf SEQ ID NO: 31Physcomitrella_patens_subsp._patens {168030621} ref|XP_001767821.1|mqqvkgrqdfqrlleaqqdlicryhthelkahqcgsillqqikvplpivwaivrsfdkpqvykriiqtckitegdggygsirevhlyssvpatcsierleilddekhiisfrvlggghrlqnyssysslheleveghpctlvlesymvdipdgntreethmfvdtvvrcnlkslaqiseqqynkdclqqkqhdqqqmyqqrhpplppipitdknmer SEQ ID NO: 32Physcomitrella_patens_subsp._patens {168028995} ref|XP_001767012.1|mrfdighndvrgfftceeehayalhsqtvelnqcgsilmqqihapievvwsivrsfgspqiykkfiqaciltvgdggvgsirevflvsgvpatssierleilddekhvfsfrvlkgghrlqnyrsyttlheqevngrqtttylesyvvdvpdgntreethmfadtvvmcnlkslaqvaewramqgitqqlstssl SEQ ID NO: 33 Vitis_vinifera {147840019}emb|CAN72620.1|mgnlyhthhllpnqcsslvvqttdaplpqvwsmvrrfdrpqsykrfvrgctlrrgkggvgsvrevnivsglpaeislerldkldddlhvmrftviggdhrlanyhstltlhedeedgvrktvvmesyvvdvpggnsagetcyfantiigfnlkalaavtetmalkanipsgfSEQ ID NO: 34 Picea_sitchensis {116785512} gb|ABK23752.1|medlsswregramwlgnppseselvcrhhrhelqgnqcssflvkhirapvhlVwsivrtfdqpqkykpfvhscsvrggitVgsirnvnVksglpataseerleilddnehvfsikilggdhrlqnyssiitVhpeiidgrpgtlViesyvvdvpegntreetrffvealvkcnlksladvserlasqhhtellert SEQ ID NO: 35 Solanum_tuberosum {78191398}gb|ABB29920.|mnangfcgvekeyirkhhlhepkenqcssflvkhirapvhlvwslvrrfdqpqkykpfisrcivqgdleigslrevdvksglpattsterlellddeehilsvrivggdhrlrnyssvisvhpevidgrpgtvvlesfvvdvpegntkdetcyfvealincnlksladiservavqdrtepidqv SEQ ID NO: 36 Medicago_truncatula {217075184} gb|ACJ85952.1|mnngceqqqysvietqyirrhhkhdlrdnqcssalvkhikapvhlvwslvrrfdqpqkykpfisrcimqgdlsigsvrevnyksglpattsterleqlddeehilgirivggdhrtrnyssiitvhpgvidgrpgtmviesfvvdvpegntkdetcyfvealirynlssladvsermavqgrtdpininp SEQ ID NO: 37 Vitis_vinifera {157358179} emb|CAO65816.1|msgygcikmedeyirrhhrheirdnqcssslvkhikapvhlvwslvrsfdqpqkykpfvsrcivqgdleigsvrevnyksglpattsterlellddeehifgmrivggdhrlknyssivtvhpeiidgrpgtlviesfvvdvpdgntkdetcyfvealikcnlksladvserlaiqdrtepidrm SEQ ID NO: 38 Vitis_vinifera {157360187} emb|CAO69376.1|mngnglssmeseyirrhhrhepaenqcssalvkhikapvplvwslvrrfdqpqkykpfisrcvvqgnleigslrevdvksglpattsterlelldddehilsmriiggdhrlrnyssiislhpeiidgrpgtmviesyvvdvpegntkdetcyfvealikcnlksladvserlavqdrtepidrm SEQ ID NO: 39 Oryza_sativa_Japonica_Group {125597584}gb|EAZ37364.1|meahveralreglteeeraalepavmahhtfppstttattaaatctslvtqrvaapvravwpivrsfgnpqrykhfvrtcalaagngpsfgsvrevtvvsgpsrlppgterlemldddrhiisfrvvggqhrlrnyrsvtsvtefqppaagpgpappycvvvesyvvdvpdgntaedtrmftdtvvklnlqmlaavaedsssasrrrd SEQ ID NO: 40 Capsicum_annuum {47558817}gb|AAT35532.1|mmnangfsgvekeyirkhhlhqpkenqcssflvkhirapvhlvwslvrrfdqpqkykpfvsrciaqgdleigslrevdvksglpattsterlellddeehilsfriiggdhrlrnyssiislhpevidgrpgtlviesfvvdvpqgntkdetcyfvealincnlksladvserlavqdrtepidqv SEQ ID NO: 41 Populus_trichocarpa {118481075} gb|ABK92491.1|mngsdaysateaqyvrrhhkheprenqctsalvkhikapahlvwslvrrfdqpqrykpfvsrcvmngelgigsvrevnvksglpattsterlellddeehilgvqivggdhrlknyssimtvhpefidgrpgtlviesfivdvpdgntkdetcyfvealircnlksladvsermavqdrvepvnqf SEQ ID NO: 42 Capsicum_annuum {104304209} gb|ABF72432.1|mnangfsgvekeyirkhhlhqpkenqcssflvkhirapvhlvwslvrrfdqpqkykpfvsrciaqgdleigslrevdvksglpattsterlellddeehilsfriiggdhrlrnyssiislhpevidgrpgtlviesfvvdvpqgntkdetcyfvealincnlksladvserlavqdrtepidqv SEQ ID NO: 43 Populus_trichocarpa_x_Populus_deltoides {118489403}gb|ABIK96505.1|mngsdaysateaqyvahhkheprenqctsalvkhikapahlvwslvrrfdqpqrykpfvsrcvmngelgigsvrevnvksglpattsterlellddeehilgvqivggdhrlknyssimtvhpefidgrpgtlviesflvdvpdgntkdetcyfvkalircnlksladvsermavqdrvepvnqf SEQ ID NO: 44 Pisum_sativum {56384584} gb|AAV85853.1|mnnggeqysaietqyirrrhkhdlrdnqcssalvkhikapvhlvwslvrrfdqpqkykpfvsrcimqgdlgigsvrevnvksglpattsterleqlddeehilgirivggdhrtrnyssvitvhpevidgrpgtmviesfvvdvpegntrdetcyfvealirgnlssladvsermavqgrtdpinvnp SEQ ID NO: 45 Vitis_vinifera {15734988} emb|CAO39744_1|meaqvicrhhaheprenqcssylvrhvkapanlvwslvrrfdqpqkykpfvsrcvvqgdlrigsvrevnvktglpattsterlelfdddehvlgikildgdhrlmyssvitvhpeiidgrpgtlviesfvvdvpegntkddtcyfvralincnlkclaevsermamlgrvepanavSEQ ID NO: 46 Vitis_vinifera {147856414} emb|CAN82501.1|mmeaqvicrhhaheprenqcssylvrhvkapanlvwslvrrfdqpqkykpfvsrcvvqgdlrigsvrevnvktglpattsterlelfdddehvlgikildgdhrlmyssvitvhpeiidgrpgtlviesfvvdvpegntkddtcyfvralincnlkclaevsermamlgrvepanav SEQ ID NO: 47 Arachish_hypogaca {196196276} gb|ACG76109.1|mmngscggggggeaygaieaqyirrhhrheprdnqctsalvkhirapvhlvwslvrrfdqpqkykpfvsrcimqgdlgigsvrevnvksglpattsterleqlddeehilgirivggdhrtrnyssiitvhpeviegrpgtmviesfvvdvpdgntkdetcxfvealircnlssladvsermavqgrtdpinq SEQ ID NO: 48 Zea_mays {195639836} gb|ACG39386.1|mvvemdggvgvaagggggaqtpapapprrwrladercdlrametdyvrrfhrheprdhqcssavakhikapvhlvwslvrrfdqpqlfkpfvsrcemkgnieigsvrevnvksglpatrsterlellddderilsvrfvggdhrlqnyssiltvhpevidgrpgtlviesfvvdvpdgntkdetcyfveallkcnlrslaevsegqvimdqtepldr SEQ ID NO: 49Zea_mays {194691986} gb|ACF80077.1|mvvemdggvgvaaaggggaqtpapppprrwrladercdlrametdyvrrfhrheprdhqcssavakhikapvhlvwslvrrfdqpqlfkpfvsrcemkgnieigsvrevnvksglpatrsterlellddderilsvrfvggdhrlqnyssiltvhpevidgrpgtlviesfvvdvpdgntkdetcyfveallkcnlrslaevsegqvimdqtepldr SEQ ID NO: 50Oryza_sativa_Japonica_Group {115468346} ref|NP_001057772.1|mngvggaggaaagklpmvshrrvqwrladercelreeemeyirrfhrhepssnqctsfaakhikaplhtvwslvrrfdqpqlfkpfvrncvmrenfiatgcirevnvqsglpatrsterlellddnehilkvnfiggdhmlknyssiltvhsevidgqlgtlvvesfivdvpegntkddisyfienvlrcnlrtladvseerlanp SEQ ID NO: 51Oryza_sativa_Indica_Group {1255555821} gb|EAZ01188.1|mngaggaggaaagklpmvshrqvqwrladercelreeemeyirqfhrhepssnqctsfvakhikaplqtvwslvrrfdqpqlfkpfvrkcvmreniiatgcvrevnvqsglpatrsterlellddnehilkvkfiggdhmlknyssiltihsevidgqlgtlvvesfvvdipegntkddicyfienilrcnlmtladvseerlanp SEQ ID NO: 52Oryza_sativa_Japonica_Group {125581525} gb|EAZ22456.1|mvevgggaaeaaagrrwrladercdlraaeteyvrrfhrheprdhqcssavakhikapvhlvwslvrrfdqpqlfkpfvsrcemkgnieigsvrevnyksglpatrsterlellddnehilsvrfvggdhrlknyssiltvhpevidgrpgtlviesfvvdvpegntkdetcyfveallkcnlkslaevserlvcqgpnrapstr SEQ ID NO: 53Oryza_sativa_Japonica_Group {115445369} ref|NP_001046464.1|mvevgggaaeaaagrrwrladercdlraaeteyvrrfhrheprdhqcssavakhikapvhlvwslvrrfdqpqlfkpfvsrcemkgnieigsvrevnvksglpatrsterlellddnehilsvrfvggdhrlknyssiltvhpevidgrpgtlviesfvvdvpegntkdetcyfveallkcnlkslaevserlvvkdqtepldr SEQ ID NO: 54 Medicago_truncatula {217075288}gb|ACJ86004.1|mekmngtenngvfnstemeyirrhhnqqpgenqcssalvkhirapvplvwslvrrfdqpqkykpfvsrcvvrgnleigslrevdvksglpattsterlevlddnehilsiriiggdhrlrnyssimslhpeiidgrpgtlviesfvvdvpegntkdetcyfvealikenlkslsdvseghavqdltepldrvhellisg SEQ ID NO: 55 Medicago_truncatula {217071196}gb|AC.183958.1|mekmngtenngvfnstemeyirrhhnqqpgenqcssalvkhirapvplvwslvrrfdqpqkykpfvsrcvvrgnleigslrevdvksglpattsterlevlddnehilsiriiggdhrlrnyssimslhpeiidgrpgtlviesfvvdvpegntkdetcyfvealikcnlkslsdvseghaaqdltepldrmhellisg SEQ ID NO: 56 Zea_mays {195625792} gb|ACG34726.1|mvglvggstaraehvvanaggeaeyvrrmhrhaptehqctstlvkhikapvhlvwqlvrrfdqpqrykpfvrncvvrgdqlevgslrdvnyktglpattsterleqldddlhilgvkfvggdhrlqnyssiitvhpesidgrpgtlviesfvvdvpdgntkdetcyfveavikcnlnslaevseqlavesptslidq SEQ ID NO: 57 Zea_mays {195608384} gb|ACG26022.1|mvglvggstaraehvvanaggeaeyvrrmhrhaptehqctstlvkhikapvhlvwelvrrfdqpqrykpfvrncvvrgdqlevgslrdvnyktglpattsterleqldddlhilgvkfvggdhrlqnyssiitvhpesidgrpgtlviesfvvdvpdgntkdetcyfveavikcnlnslaevseqlavesptslidq SEQ ID NO: 58 Zea_mays {194704156} gb|ACF86162.1|mvmvemdggvgggggggqtpaprrwrladercdlrametdyvrrfhrheprehqcssavakhikapvhlvwslvrrfdqpqlfkpfvsrcemkgnieigsvrevnyksglpatrsterlellddnehilsvrfvggdhrlqnyssiltvhpevidgrpgtlviesfvvdvpdgntkdetcyfveallkcnlkslaevserqvvkdqtepldr SEQ ID NO: 59Oryza_sativa_Japonica_Group {115468344} ref|NP_001057771.1|mngaggaggaaagklpmvshrrvqcrladkrcelreeemeyirqfhrhepssnqctsfvakhikaplqtvwslvrrfdqpqlfkpfvrkcvmreniivtgcvrevnvqsglpatrsterlellddnehilkvkfiggdhmlknyssiltihsevidgqlgtlvvesfvvdipdgntkddicyfienvlrcnlmtladvseerlanp SEQ ID NO: 60 Zea_mays {194701978}gb|ACF85073.1|mvglvggstaraehvvanaggeteyvrrlhrhapaehqctstlvkhikapvhlvwelvrsfdqpqrykpfvrncvvrgdqlevgslrdvnyktglpattsterleqldddlhilgvkfvggdhrlqnyssiitvhpesidgrpgtlviesfvvdvpdgntkdetcyfveavikcnlkslaevseqlavesptspidq SEQ ID NO: 61 Oryza_sativa_Indica_Group {125555585}gb|EAZ01191.1|mngvggaggaaagklpmvshrrvqwrladercelreeemeyirrfhrhepssnqctsfaakhikaplhtvwslvrrfdqpqlfkpfvrncvmreniiatgcirevnvqsglpatrsterlellddnehilkvkfiggdhmlknyssiltvhsevidgqlgtlvvesfivdvlegntkddisyfienvlrcnlrtladvseerlanp SEQ ID NO: 62Oryza_sativa_Japonica_Group {115462647} ref|NP_00105492.1|mvglvggggwrvgddaaggggggavaagaaaaaeaehmrrlhshapgehqcssalvkhikapvhlvwslvrsfdqpqrykpfvsrcvvrggdleigsvrevnyktglpattsterlelldddehilsvkfvggdhrtrnyssivtvhpesidgrpgtlviesfvvdvpdgntkdetcyfveavikcnltslaevserlavqsptspleq SEQ ID NO: 63Oryza_sativa_Japonica_Group {50251668} dbj|BAD29692.1|mvemdaggrpepsppsgqcssavtmrinapvhlvwsivrrfeephifqpfvrgctmrgstslavgcvrevdfksgfpakssverleilddkehvfgvriiggdhrlknyssvltakpevidgepatlvsesfvvdvpegntadetrhfveflircnlrslamvsqrlllaqgdlaeppaq SEQ ID NO: 64 Vitis_vinifera {147797548} emb|CAN64668.1|mngnglssmeseyirrhhrhepaenqcssalvkhikapvplvwslvrrfdqpqkykpfisrcvvqgnleigslrevdvksglpattsterlelldddehilsmriiggdhrlrnyssiislhpeiidgrpgtmviesyvvdvpegntkdetcyfsladvserlavagtvtepidrmSEQ ID NO: 65 Oryza_sativa_Indica_Group {218190432} gb|EEC72859.1|mvemdaggrpepsppsgqcssavtmrinapvhlvwsivrrfeephifqpfvrgctmrgstslavgcvrevdfksgfsakssverleilddkehvfgvriiggdhrlknyssvltakpevidgepatlvsesfvidvpegntadetrhfveflircnlrslamvsqrlllaqgdlaeppaq SEQ ID NO: 66 Oryza_sativa_Japonica_Group {125585934} gb|EAZ26598.1|mpcipasspgiphqhqhqhhralagvgmavgcaaeaavaaagvagtrcgahdgevpmevarhhehaepgsgrccsavvqhvaapaaavwsvvrrfdqpqaykrfvrscallagdgglgkvrerleilddeshvlsfrvvggehrlknylsvttvhpspsaptaatvvvesyvvdvppgntpedtrvfvdtivkcnlqslaktaeklaagaraags SEQ ID NO: 67Rheum_austrate {197312913} gb|ACH63237.1|mngdgyggseeefvkryhehvladhqcssvlvehinaplhlvwslvrsfdqpqkykpfvsrcvvqggdleigsvrevdvksglpattsmeelellddkehvlrvkfvggdhrlknyssivslhpeiiggrsgtmviesfivdiadgntkeetcyfieslincnlkslscvserlavediaeriaqm SEQ ID NO: 68 Oryza_saliva_Japonica_Group {125593228}gb|EAZ33287.1|mvglvggggwrvgddaaggggggavaagaaaaaeaehmrrlhsqgprrapvqlrarqahqgscsppriecanfavflaardpkivwslvrsfdqpqrykpfvsrcvvrggdleigsvrevnvktglpattsterlelldddehilsvkfvggdhrtrnyssivtvhpesidgrpgtlviesfvvdvpdgntkdetcyfveavikcnltslaemvrmislvlpfmlvdrmsgitceshlettlvrcgeyavlahvSEQ ID NO: 69 Oryza_sativa_Japonica_Group {125581370} gb|EAZ22301.1|mephmeralreavaseaerrelegvvrahhtgwnaplaavwphrarvrptrsgtstsssrassppgdgatvgsvrevavvsglpaststerleildddrhvlsfrvvggdhrlmyrsvtsvtefsspsspprpycvvvesyvvdvpegnteedtrmftdtvvklnlqklaavatsssppaagnhh SEQ ID NO: 70 Oryza_sativa_Japonica_Group {125581524}gb|EAZ22455.1|mevvwsivrrfeephifqpfvrgctmrgstslavgcvrevdfksgfpakssverleilddkehvfgvriiggdhrlknyssvltakpevidgepatlvsesfvvdvpegntadetrhfveflircnlrslamvsqrlllaqgdlaeppgqSEQ ID NO: 71 Oryza_sativa_Japonica_Group {125594587} gb|EAZ34646.1|mpytaprpsppqhsriggcggggvlkaagaaghaascvavpaevarhhehaagvgqccsavvqaiaapvdavwrtstssgaaaswtatatagplpvgsvrefrvlsglpgtssrerleildderrvlsfrvvggehrlsnyrsvttvhetaagaaaavvvesyvvdvphgntadetrmfvdtivrcnlqslartaeqlalaapraa SEQ ID NO: 72Vitis_vinifera {147770961} emb|CAN76441.1|mpisslpfslytvtpnplklitthahaftphthiftlkfmshtycphihhitsihythllxpiphmplqpplpphpilpsmpafqhlystnqhlqvalfsargpnirdfnfqdadllkldilapgsliwaawspngtdeanyvgegsptvamiakrgprhgkymafcxmyrdnvapkgvnxavatvktkrtiqlktsleiachyaginisgingevmpgqweyqvgpgqcssllaqrvhvplsavgsvvhrfdkpqryqhvikscriedgfemrmgxlrdvniisglptatntgrldmqdderhvtrcphqrqseskytennnsdassikspingpsehlktaaspktesiividtskflneedfegkdetsssnqvqiedenwetrfpntdagiw SEQ ID NO: 73Vitis_vinifera {147828564} emb|CAN59881.1|mpsaxksstvplslxqfklglrhghrvipwgdldslamlqrqldvdilvtghthrftaykheggvvinpgsatgafgsitydvnpsfvlmdidglrvvvcvyelidetaniikelharkisfgtksmixclllkrrstpkfrrkklflfqcrvqmtltltnlavsgiaqtlqvdqwtvcalifmtrrdihldkarfldfkdmgklladasglrkalsggxvtagmaifdtmrhirpdvptvcvglaavamiakrgprhgkymafcpmyrdnvapkgvnvavvtvktkrtiqlktsleiachyaginisgingevmpgqweyqvgpgqcssllaqrvhvplsavgsvvhrfdkpqryqhvikscriedgfemrmgrlrdvniisglptatntgrldmqddexhvtrcphqrqseskytennnsdassykspingpsehlktaax SEQ ID NO: 74 Oryza_sativa_Japonica_Group {149392053} gb|ABR25904.1|eigsvrevnvktglpattsterlelldddehilsvkfvggdhrlrnyssivtvhpesidgrpgtlviesfvvdvpdgntkdetcyfveavikcnl SEQ ID NO: 75 Zea_mays {194701080} gb|ACF84624.1|mvvemdggvgvaaaggggaqtpapppprrwrladercdlrametdyvrrfhrheprdhqcssavakhikapvhlvwslvrrfdqpqlfkpfvsrcemkgnieigsvrevnvksglpatrsterlellddderilsvrfvggdhrlqvcsvlhlsifcaaharyfahhlkcvleflcqmhldv1pcddaile SEQ ID NO: 76 Oryza_sativa_Japonica_Group {125597418}gb|EAZ37198.1|mngctggaggvaagrlpayslqqaqwklvdercelreeemeyvrrfhrheigsnqcnsfiakhvraplqnvwslvrrfdqpqiykpfvrkcvmrgnvetgsvreiivqsglpatrsierleflddneyilrvkfiggdhmlkkripkktyaissrtcsdsaiiavgqsncapeitamnggvsiqpwlillaffsspsnqtnpdslrdmhpgswfqillvlamftcskgsvlppsekvnvSEQ ID NO: 91 GxxRxVxxxSGxPAxxSxExLxxxDxxxxxxxxxxxGGxHRLxNYKSxxxSEQ ID NO: 92 xxxxxxxxxESxxVDxPxGNxxxxTxxFxxxxxxxNLxxLx SEQ ID NO: 93CxSxxxxxxxAPxxxxWxxxxxFxxPxxxxxFxxxC SEQ ID NO: 94GxxRxVxxxSxxPAxxSxExLxxxD SEQ ID NO: 95 GGxHRLxNYxS SEQ ID NO: 96CxSxxxxxxxAPxxxxWxxxxxFxxPxxxKxFxxxC SEQ ID NO: 97GxxRxVxxxSxLPAxxSxExLxxxD SEQ ID NO: 98 GGxHRLxNYxS SEQ ID NO: 99ESxxVDxPxGNxxxxTxxFxxxxxxxNLxxL SEQ ID NO: 100HxxxxxxxxCxSxxxxxxxAPxxxxWxxxxxFxxPxxYKxFxxxC SEQ ID NO: 101VGRxVxVxSGLPAxxSxExLxxxDxxxxxxxFxxxGGxHRLxNYxSVT SEQ ID NO: 102VxESYxVDxPxGNxxxxTxxFxDxxxxxNLQxL SEQ ID NO: 103HxHxxxxxQCxSxLVKxIxAPxHxVWSxVRRFDxPQKYKPFxSRCxVxGx SEQ ID NO: 104ExGxxREVxxKSGLPATxSTExLExLDDxEHILxIxIxGGDHRLKNYSSxxxxHxExIxGxxGTxSEQ ID NO: 105 xxESFVVDVPxGNTKxxTCxFVExLIxCNLxSLAxxxERL SEQ ID NO: 106CxSxxVxTIxAPLxLVWSILRxFDxPxxxxxFVKxCxxxSGxGG SEQ ID NO: 107GSVRxVTxVSxxPAxFSxERLxELDDESHVMxxSIIGGxHRLVNYxSKT SEQ ID NO: 108MEPHMESALRQGLSEAEQRELEGVVRAHHTFPGRAPGTCTSLVTQRVDAPLAAVWPIVRGFGSPQRYKHFIKSCDLKAGDGATVGSVREVTVVSGLPASTSTERLEILDDHRHILSFRVVGGDHRLRNYRSVTSVTEFQPGPYCVVLESYVVDVPDGNTEEDTRMFTDTVVKLNLQKLAAIATSSSAN SEQ ID NO: 109MDQQGAGGDVEVPAGLGLTAAEYEQLRPTVDAHHRYAVGEGQCSSLLAQRIHAPPAAVWAIVRRFDCPQVYKHFIRSCAVRPDPDAGDALRPGRLREVCVISGLPASTSTERLDHLDDAARVFGFSITGGEHRLRNYRSVTTVSELAGPGICTVVLESYAVDVPDGNTEDDTRLFADTVIRLNLQKLKSVAEASTSSSAPPPPSE SEQ ID NO: 110MPCIQASSPGGMPHQHGRGRVLGGGVGCAAEVAAAVAASAGGMRCGAHDGEVPAEAARHHEHAAAGPGRCCSAVVQHVAAPAAAVWSVVRRFDQPQVYKRFVRSCALLAGDGGVGTLREVRVVSGLPAASSRERLEVLDDESHVLSFRVVGGEHRLRNYLSVTTVHPSPAAPDAATVVVESYVVDVPPGNTPEDTRVFVDTIVKCNLQSLATTAEKLAAV SEQ ID NO: 111MEKAESSASTSEPDSDENHHRHPTNHHINPPSGLTPLEFASLIPSVAEHHSYLVGSGQCSSLLAQRVQAPPDAVWSVVRRFDKPQTYKHFIKSCAVKEPFHMAVGVTRDVNVISGLPAATSTERLDLLDDIRCVTGFSIIGGEHRLRNYRSVTTVHSFEDDADDGKIYTVVLESYVVDVPDGNTEEDTRLFADTVVKLNLQKLASVTEGTNRDGDGKSHSR SEQ ID NO: 112MEKTHSSSAEEQDPTRRHLDPPPGLTAEEFEDLKPSVLEHHTYSVTPTRQSSSLLAQRIHAPPHAVWSVVRCFDNPQAYKHFIKSCHVKEGFQLAVGSTRDVHVISGLPAATSTERLDLLDDDRHVIGFTIVGGDHRLRNYRSVTSVHGFECDGKIWTVVLESYVVDVPEGNTEEDTRLFADTVVKLNLQKLASVSEGMCGDGDGDGDGKGNKS SEQ ID NO: 113MLQNSSMSSLLLHRINGGGGATTATNCHDTVFMTVPDGVARYHTHAVAPNQCCSSVAQEIGASVATVWSVLRRFDNPQAYKHFVKSCHVIGGDGDVGTLREVHVISGLPAARSTERLEILDDERHVISFSVVGGDHRLANYRSVTTLHPTASSASGGCSGTVVVESYVVDVPPGNTREDTRVFVDTIVKCNLQSLAQTAENLTLRDYKCCS SEQ ID NO: 114MTSLQFHRFNPATDTSTAIANGVNCPKPPSTLRLLAKVSLSVPETVARHHAHPVGPNQCCSVVIQAIDAPVSAVWPVVRRFDNPQAYKHFVKSCHVVAAAGGGEDGIRVGALREVRVVSGLPAVSSTERLEILDDERHVMSFSVVGGDHRLRNYRSVTTLHGDGNGGTVVIESYVVDVPPGNTKEETCVFVDTIVRCNLQSLAQIAET SEQ ID NO: 115AYPVLGLTPEEFSELESIINTHHKFEPSPEICSSIIAQRIDAPAHTVWPLVRSFENPQKYKHFVKSCNMRSGDGGVGSIREVTVVSGLPASTSTERLEILDDDKHLLSFRVVGGEHRLHNYRSVTSVNEFKNPDNGKVYTIVLESYVVDIPEGNTGVDTKMFVDTVVKLNLQKLGE SEQ ID NO: 116EFTELESTINTHHKFEASPEICSSIIAQRIDAPAHTVWPLVRSFENPQKYKHFVKSCNMRSGDGGVGSIREVTVVSGLPASTSTERLEILDDDNHLLSFRVVGGEHRLHNYRSVTSVNEFKRPDNGKVYTIVLESYVVDIPEGNTGVDTKMFVDTVVKLNLQKLGEVAMATN SEQ ID NO: 117MTELSSREVEYIRRHHSKAAEDNQCASALVKHIRAPLPLVWSLVRRFDEPQKYKPFVSRCVVRGNLEIGSLREVDVKSGLPATTSTERLEILDDNHHILSVRIIGGDHRLRNYSSIMSLHPEIVDGRPGTLVIESFVVDIPEGNTKDETCYFVEALIKCNLKSLADVSEGLTLQDHTEPIDRKYELLITRG SEQ ID NO: 118MNGGESYGAIETQYIRRHHKHEPRENQCTSALVKHIRAPVHLVWSLVRRFDQPQKYKPFVSRCIMQGDLGIGSVREVNVKSGLPATTSTERLEQLDDEEHILGIRIVGGDHRLRNYSSIITVHPEVIDGRPGTMVIESFVVDVPDGNTRDETCYFVEALIRCNLSSLADVSERMAVQ GRTNPINHSEQ ID NO: 119 MSPNNPSTIVSDAVARHHTHVVSPHQCCSAVVQEIAAPVSTVWSVVRRFDNPQAYKHFVKSCHVILGDGDVGTLREVRVISGLPAAVSTERLDVLDDERHVIGFSMVGGDHRLSNYRSVTILHPRSATDTVVVESYVVDVPAGNTTEDTRVFVDTILRCNLQSLAKFAENLTN KLHQRSEQ ID NO: 120MSRSHNKRKPFSFIFKITLLELLSSLLSSSLRFAMDKTHSGEEQDPNPTHPTRNHLDPPPGLTPEEFEDLKPSVLEHHTYSVTPTRQCSSLLAQRIHAPPHTVWTVVRCFDNPQAYKHFIKSCHVKEGFQLAVGSTRDVHVISGLPAATSTERLDLLDDDRHVIGFTIVGGDHRLRNYRSVTSVHGFERDGKIWTVVLESYVVDVPEGNTEEDTRLFADTVVKLNLQKLASVTEG MCGDSDGKGNNSEQ ID NO: 121MEKAESSASTSEPDSDDNHHRHPTNHHLNPPSGLTPLEFASLVPSVAEHHSYLVGPGQCSSLLAQRVHAPPDAVWSFVRRFDKPQTYKHFIKSCAVKEPFHMAVGVTRDVNVISGLPAATSTERLDFLDDVRRVTGFSIIGGEHRLRNYRSVTTVHSFDDDNASADGKIYTVVLESYVVDVPDGNTEEDTRLFADTVVKLNLQKLASVTEGTNGDGDGKPHSR SEQ ID NO: 122MPSSLHFDRFNPITHAATTVAIANGVNCPKQPQAPPSSTAARRLVVPSLSSGRGIAAPDTVALHHAHVVDPNQCCSIVTQHINAPVSAVWAVVRRFDNPQGYKNFVRSCHVITGDGIRVGAVREVRVVSGLPAETSTERLEILDDERHVISFSMVGGDHRLRNYQSVTTLHANGNGTLVIESYVVDVPQGNTKEETCVFVDTIVRCNLQSLAQIAENRTNNCEHTAQHC SEQ ID NO: 123MNGIGNDGGGGLSNVEMEYIRRHHRHEPGENQCGSALVKHIRAPVPQVWSLVRRFDQPQKYKPFVSRCVVRGNLEIGSLREVDVKSGLPATTSTERLELLDDNEHLLSIRIIGGDHRLRNYSSIMSLHPEIIDGRPGTLVIESFVVDVPEGNTKDETCYFVEALIKCNLKSLADVSEGIAVQDRTEPIDRI SEQ ID NO: 124MVARHHAHAVGPNQCCSFVIQAIDAPVSAVWPVVRRFDNPQAYKHFVKSCHVVAAGGAGGDGGIHVGALREVRVVSGLPAVSSTERLEILDDERHVMSFSVVGGDHRLRNYRSVTTLHGDGSNGGTVVIESYVVDIPAGNTKEETCVFVDTIVRCNLQSLAQMAENMGS SEQ ID NO: 125MTILPHSNNKSSNHKFIAHQNYMASETHHHVQGLTPEELTKLEPIIKKYHLFEQSPNTCFSIITYRIEAPAKAVWPFVRSFDNPQKYKHFIKGCNMRGDGGVGSIREVTVVSGLPASTSTERLEILDDDKHVLSFRVVGGEHRLKNYRSVTSVNEFNKEGKVYTIVLESYIVDIPEGNTEEDTKMFVDTVVKLNLQKLGVVAMASSMHGQ SEQ ID NO: 126MNRIGNGGGGGGGLSNVEMEYIRRHHRHEPGENQCGSALVKHIRAPVPQVWSLVRRFDQPQKYKPFISRCVVRGNLEIGSLREVDVKSGLPATTSTERLELLDDNEHILSIRIIGGDHRLRNYSSIMSLHPEIIDGRPGTLVIESFVVDVPEGNTKDETCYFVEALIKCNLKSLADVSEGLAVQDCTEPIDRI SEQ ID NO: 127MASETHHHVQGLTPEELTQLEPIIKKYHLFEASSNKCFSIITHRIEAPASSVWPLVRNFDNPQKYKHFIKGCNMKGDGSVGSIREVTVVSGLPASTSTERLEILDDDKHVLSFRVVGGEHRLQNYRSVTSVNEFHKEGKVYTIVLESYIVDIPEGNTEEDTKMFVDTVVKLNLQKL GVVAMASSMNGRSEQ ID NO: 128 MLPNNPSTIVPDAVARHHTHVVSPQQCCSAVVQEIAAPVSTVWSVVRRFDNPQAYKHFVKSCHVILGDGDVGTLREVHVISGLPAAVSTERLDVLDDERHVIGFSMVGGDHRLFNYRSVTTLHPRSAAGTVVVESYVVDVPPGNTTEDTRVFVDTILRCNLQSLAKFAENLTK LHQRSEQ ID NO: 129MNGGESYGAIETQYIRRHHKHEPRENQCTSALVKHIRAPVHLVWSLVRRFDQPQKYKPFVSRCIMQGDLGIGSVREVNVKSGLPATTSTERLEQLDDEEHILGIRIVGGDHRLRNYSSIITVHPEVIDGRPGTMVIESFVVDVPDGNTRDETCYFVEALIRCNLSSLADVSERMAVQ GRTNPINHSEQ ID NO: 130MGITIGIQCLEIEEISICDGMFCYLVDFVDVKEKMNYCLMWFGYFPSQVWSLVRRFDQPQKYKPFVSRCIMQGDLGIGSVREVNVKSGLPATTSTERLEQLDDEEHILGIRIVGGDHRLRNYSSIITVHPEVIDGRPSTMVIESFVVDVPDGNTRDETCYFVEALIRCNLSSLADVSERMAVQGRTDPINH SEQ ID NO: 131MNGGESYGAIETQYIRRHHKHEPRENQCTSALVKHIRAPVHLVWSLVRRFDQPQKYKPFVSRCIMQGDLGIGSVREVNVKSGLPATTSTERLEQLDDEEHILGIRIVGGDHRLRNYSSIITVHPEVIDGRPSTMVIESFVVDVPDGNTRDETCYFVEALIRCNLSSLADVSERMAVQG RTDPINHSEQ ID NO: 132METHVERALRATLTEAEVRALEPAVREHHTFPAGRVAAGTTTPTPTTCTSLVAQRVSAPVRAVWPIVRSFGNPQRYKHFVRTCALAAGDGASVGSVREVTVVSGLPASSSTERLEVLDDDRHILSFRVVGGDHRLRNYRSVTSVTEFQPGPYCVVVESYAVDVPEGNTAEDTRMFTDTVVRLNLQKLAAVAEESAAAAAAGNRR SEQ ID NO: 133MEPHMETALRQGGLSELEQRELEPVVRAHHTFPGRSPGTTCTSLVTQRVDAPLSAVWPIVRGFAAPQRYKHFIKSCDLRSGDGATVGSVREVTVVSGLPASTSTERLEILDDDRHILSFRVVGGDHRLRNYRSVTSVTEFHHHHQAAAGRPYCVVVESYVVDVPEGNTEEDTRMFTDTVVKLNLQKLAAIATSSAAAAASNSST SEQ ID NO: 134MVESPNPNSPSRPLCIKYTRAPARHFSPPLPFSSLIISANPIEPKAMDKQGAGGDVEVPAGLGLTAAEYEQLRSTVDAHHRYAVGEGQCSSLLAQRIQAPPAAVWAIVRRFDCPQVYKHFIRSCALRPDPEAGDALRPGRLREVSVISGLPASTSTERLDLLDDAARVFGFSITGGEHRLRNYRSVTTVSELADPGICTVVLESYVVDVPDGNTEDDTRLFADTVIRLNLQKLKSVAEANAAAAASFVSVVPPPEPEE SEQ ID NO: 135MPCLQASSSPGSMPHQHHGRVLAGVGCAAEVAAAAVAATSPAAGMRCGAHDGEVPAEAARHHEHAAPGPGRCCSAVVQHVAAPASAVWSVVRRFDQPQAYKRFVRSCALLAGDGGVGTLREVRVVSGLPAASSRERLEVLDDESHVLSFRVVGGEHRLQNYLSVTTVHPSPAAPDAATVVVESYVVDVPPGNTPEDTRVFVDTIVKCNLQSLATTAEKLAAV SEQ ID NO: 136MVEMDGGVGVVGGGQQTPAPRRWRLADELRCDLRAMETDYVRRFHRHEPRDHQCSSAVAKHIKAPVHLVWSLVRRFDQPQLFKPFVSRCEMKGNIEIGSVREVNVKSGLPATRSTERLELLDDNEHILSVKFVGGDHRLQNYSSILTVHPEVIDGRPGTLVIESFVVDVPDGNTKDETCYFVEALLKCNLKSLAEVSERQVIKDQTEPLDR SEQ ID NO: 137MPYTAPRP SPQQHSRVTGGGAKAAIVAASHGASCAAVPAEVARHHEHAARAGQCCSAVVQAIAAPVGAVWSVVRRFDRPQAYKHFIRSCRLVDDGGGGAGAGAGATVAVGSVREVRVVSGLPATSSRERLEILDDERRVLSFRVVGGEHRLANYRSVTTVHEAEAGAGGTVVVESYVVDVPPGNTADETRVFVDTIVRCNLQSLARTAERLALALA SEQ ID NO: 138EXLXXXDXXXXXXXXXXXGGXHXL

What is claimed is:
 1. An agricultural chemical formulation formulatedfor contacting to plants, the formulation comprising a compound selectedfrom the following formulas:

wherein R¹ is selected from the group consisting of aryl and heteroaryl,optionally substituted with 1-3 R^(1a) groups; each R^(1a) isindependently selected from the group consisting of H, halogen, C₁₋₆alkyl, C₁₋₆ haloalkyl, C₂₋₆ alkenyl, C₂₋₆ alkynyl, C₁₋₆ alkoxy, C₁₋₆haloalkoxy, C₁₋₆ hydroxyalkyl, —NR′R″, —SR′, —OH, —CN, —NO₂, —C(O)R′,—C(O)OR′, —C(O)NR′R″, —N(R′)C(O)R″, —N(R′)C(O)OR″, —N(R′)C(O)NR′R″,—OP(O)(OR′)₂, —S(O)₂OR′, —S(O)₂NR′R″, cycloalkyl, heterocycloalkyl, aryland heteroaryl, wherein the aryl group is optionally substituted with—NO₂ and the heteroaryl group is optionally substituted with C₁₋₆ alkyl;alternatively, adjacent R^(1a) groups can combine to form a memberselected from the group consisting of cycloalkyl, heterocycloalkyl, aryland heteroaryl, wherein the aryl group is optionally substituted with—OH; R′ and R″ are each independently selected from the group consistingof H and C₁₋₆ alkyl; R² is selected from the group consisting of C₂₋₆alkenyl, cycloalkenyl, aryl and heteroaryl, wherein the aryl orheteroaryl is optionally substituted with 1-3 R^(1a) groups; R³ is H oris optionally combined with R² and the atoms to which each is attachedto form a heterocycloalkyl optionally substituted with 1-3 R^(1a)groups; R⁴ is a heteroaryl, optionally substituted with 1-3 R^(1a)groups; R⁵ is selected from the group consisting of C₁₋₆ alkyl and aryl,wherein the aryl is optionally substituted with 1-3 R^(1a) groups; eachof R⁶ and R⁷ are independently selected from the group consisting ofaryl and heteroaryl, each optionally substituted with 1-3 R^(1a) groups;R⁸ is selected from the group consisting of cycloalkyl and aryl, eachoptionally substituted with 1-3 R^(1a) groups; R⁹ is H or is optionallycombined with a R^(1a) group of R⁸ and the atoms to which each isattached to form a heterocycloalkyl; subscript n is 0-2; X is absent oris selected from the group consisting of —O—, and —N(R′)—; Y is absentor is selected from the group consisting of —C(O)— and —C(R′,R″)—; Z isabsent or is selected from the group consisting of —N═, and—C(S)—N(R′)—, such that one of Y and Z is absent; each of R¹⁰ and R¹¹are independently selected from the group consisting of H, C₁₋₆ alkyl,—C(O)OR′, and C₁₋₆ alkenyl-C(O)OH, wherein at least two of the R¹⁰ andR¹¹ groups are C₁₋₆ alkyl and at least one of the R¹⁰ and R¹¹ groups isC₁₋₆ alkenyl-C(O)OH; alternatively, two R¹⁰ or R¹¹ groups attached tothe same carbon are combined to form ═O; alternatively, one R¹⁰ groupand one R¹¹ group are combined to form a cycloalkyl having from 3 to 6ring members; each of subscripts k and m is an integer from 1 to 3, suchthat the sum of k and m is from 3 to 4; each of subscripts p and r is aninteger from 1 to 10; wherein two of the R¹⁰ and R¹¹ groups on adjacentcarbons are combined to form a bond; R¹² is a C₁₋₆ alkyl, substitutedwith a ═O; R¹³ is C₁₋₆ alkenyl-C(O)OH; R¹⁴ is selected from the groupconsisting of H and C₁₋₆ alkyl; and subscript r is an integer from 1 to10; with the proviso that when R¹ is 4-bromo-naphthalen-1-yl, and n is1, R² is other than unsubstituted pyrid-2-yl.
 2. The formulation ofclaim 1, wherein the compound is a compound depicted in FIG. 8 or FIG.12.
 3. The formulation of claim 1, wherein the compound is a compound ofFormula (I).
 4. The formulation of claim 3, wherein the compound is acompound selected from:


5. The formulation of claim 1, further comprising at least one of anherbicide, fungicide, pesticide, or fertilizer.
 6. The formulation ofclaim 1, further comprising a surfactant.
 7. The formulation of claim 1,further comprising a carrier.
 8. A method of increasing stress tolerancein a plant, the method comprising contacting the plant with a sufficientamount of the formulation of claim 1 to increase stress tolerance in theplant compared to not contacting the plant with the formulation.
 9. Themethod of claim 8, wherein the plant is a monocot.
 10. The method ofclaim 8, wherein the plant is a dicot.
 11. The method of claim 8,wherein the plant is a seed.
 12. The method of claim 8, wherein thestress tolerance is drought tolerance.
 13. The method of claim 8,wherein the contacting step comprises delivering the formulation to theplant by aircraft or irrigation.
 14. A method of inhibiting seedgermination in a plant, the method comprising contacting a seed with asufficient amount of the formulation of claim 1 to inhibit germination.